灰盖鬼伞过氧化物酶功能表达及部分酶学特性

摘要:【目的】旨在用毕赤酵母高效表达灰盖鬼伞过氧化物酶。【方法】借助DNAworks 3.1软件设计、优化引物,用自己构建的基因合成、定点突变平台合成了毕赤酵母密码子偏好性的灰盖鬼伞过氧化物酶基因,测序后构建在表达载体pPICZαA上,整合于巴斯德毕赤酵母GS115染色体,来自酿酒酵母的α因子作为信号肽序列指导重组蛋白的分泌表达。从82个PCR检测为阳性的酵母转化子中筛选出6株高Zeocin抗性的菌进行表达,选表达酶活性最高的作为实验菌株命名为CIP/GS115。【结果】以ABTS为底物时,CIP/GS115 在甲醇诱导第4天酶活最高达到487.5 U/mL,是目前摇瓶培养诱导表达灰盖鬼伞过氧化物酶活性最高报道。纯化后的酶最适反应温度为25℃,45℃酶反应速度是最适温度时的61.5%,在低于40℃时比较稳定,超过45℃稳定性迅速下降。最适反应pH 为5.0,在pH 4.5－6.5之间比较稳定。以不同的底物研究纯酶底物特异性发现最适底物的顺序是:ABTS ＞ 愈创木酚＞ 2,6-二甲氧苯酚＞ 2,4-二氯苯酚＞ 苯酚。【结论】灰盖鬼伞过氧化物酶在毕赤酵母中的高效分泌表达和高的特殊活性为该酶在废水处理、染料脱色等方面的工业化应用奠定了一定基础。     

共表达分子伴侣PDI和Ero1对灰盖鬼伞过氧化物酶在毕赤酵母中表达的影响

来源于灰盖鬼伞长度为1 092 bp的CiP目的基因与AOX1启动子一起整合进酵母染色体基因组中。重组蛋白CiP在酿酒酵母信号肽的引导下成功分泌到胞外,质谱鉴定为目的蛋白,成功在毕赤酵母中表达灰盖鬼伞过氧化物酶(CiP)。将伴侣蛋白内质网氧化还原酶1(Ero1)、二硫键异构酶(PDI)分别单独及同时转入CiP酵母受体菌中,研究它们对CiP在毕赤酵母中表达的影响。结果表明:在摇瓶中,相对于无分子伴侣的菌株,单独整合PDI及同时整合Ero1、PDI菌株的CiP酶活分别提高了2.43和2.62倍,活力达到316 U/m L和340 U/m L。挑选同时整合Ero1、PDI伴侣蛋白的CiP菌株,5 L发酵罐进行高密度发酵,酶活最高达到3 379 U/m L,比摇瓶提高约10倍。本实验结果较目前已报道的1 200 U/m L已是最高水平。     

环腺苷酸在灰盖鬼伞子实体发育中的效用研究

通过测试ｃＡＭＰ,子实体浸提液对灰盖鬼伞（Ｃｏｐｒｉｎｕｓ　ｃｉｎｅｒｅｕｓ）双核体菌丝的子实体诱导测试研究及通过用腺苷分别抑制ｃＡＭＰ在子实体发育各阶段的合成,对ｃＡＭＰ在灰盖克伞子实体发育中的影响进行了研究。结果表明：ｃＡＭＰ及子实体浸提液对灰盖鬼伞双核体菌丝的子实体形成并无诱导信号作用,阻止ｃＡＭＰ的合成延缓了子实体原基的发生,菌盖的形成,担孢子梗的形成,也延缓了质配和担孢子的发生,但菌柄的延长,子实体的成熟,担孢子的着色与释放则不受ｃＡＭＰ合成受阻的影响。     

牻牛儿基牻牛儿基焦磷酸合酶和紫杉二烯合酶基因在灰盖鬼伞中的组合表达

紫杉二烯是紫杉醇合成途径中的前体物质。紫杉醇是红豆杉的一种重要的次级代谢产物,是一种重要的新型抗癌药物。然而,紫杉醇在植物中含量低且难提取,限制了高效应用。利用基因工程手段,借助担子菌类真菌灰盖鬼伞具有的内源类异戊二烯合成途径,构建含有牻牛儿基牻牛儿基焦磷酸(Geranylgeranyl diphosphate,GGPP)合酶和紫杉二烯合酶的融合基因表达载体p Bg GGTS和独立表达盒表达载体p Bg GGg TS,并分别转入灰盖鬼伞LT2菌株中,经过选择性筛选、PCR鉴定、Southern blotting杂交验证,分别获得了5株融合表达的灰盖鬼伞工程菌和5株独立表达盒的灰盖鬼伞工程菌株。各随机挑选了1株工程菌株,分别提取菌丝体和发酵液分析。GC-MS分析表明,两种工程菌株与原出发菌株的菌丝提取物无明显差异峰,而与出发菌株的发酵液提取物相比,两种转基因灰盖鬼伞的发酵液中均出现了明显的差异峰,采用GC-MS特征质量离子分析方法判定为紫杉二烯,分别为44 ng/L(转化p Bg GGg TS)和30 ng/L(转化p Bg GGTS)。结果表明,通过在灰盖鬼伞融合基因或各自独立表达的形式共表达ggpps和ts基因,可以生物合成紫杉二烯。     

细菌脱色酶TpmD的酶学特性研究

从嗜水气单胞菌DN322中分离纯化出能够对三苯基甲烷类染料结晶紫、碱性品红、灿烂绿及孔雀绿进行高效脱色的脱色酶,命名为TpmD。在测定TpmD分子量、等电点及对不同三苯基甲烷染料脱色的动力学参数、脱色过程对分子氧及NADH/NADPH具有依赖性的基础上,又进一步从黄素FAD/FMN对酶活力的影响、酶抑制剂、酶蛋白N-末端测序及酶溶液的特征吸收光谱等方面对TpmD的酶学本质进行了分析。结果表明,TpmD不含核黄素,其脱色活性也不因加入FAD或FMN而提高。TpmD的N-末端氨基酸序列与多种氧化还原酶具有同源性。甲吡酮及维生素C(Vc)对TpmD的脱色活性具有明显的抑制作用。TpmD酶蛋白的溶液在408nm处有一特征吸收峰,但在连二亚硫酸钠的还原条件下通入CO气体后,该酶却不具有P450酶在450nm处的特征吸收峰。上述结果显示脱色酶TpmD是一种新的氧化酶。     

耐过氧化氢的锰过氧化物酶对三苯甲烷类染料的脱色

[目的]从一株白腐菌Trametes sp.SQ01中获得一种新型的锰过氧化物酶,探讨该酶的底物特异性和对过氧化氢的耐性,以及其对三苯甲烷类染料的脱色能力.[方法]通过丙酮沉淀和DEAE-cellulose 52柱层析法纯化锰过氧化物酶.利用UV-2010紫外可见分光光度法研究锰过氧化物酶对过氧化氢的耐性,同时,用紫外可见分光光度计对三苯甲烷类染料脱色效果进行分析.[结果]通过两步纯化,获得了均一性的锰过氧化物酶.该酶的最适pH和温度分别是4.5和70℃,在pH 3.0-8.0时,酶活相对稳定.该酶在二价锰离子存在下能够氧化2,6-二甲氧基苯酚、愈创木酚、2,2＇-连氮-双-(3-乙基苯并噻唑啉磺酸)和过氧化氢等化合物,同时也能作用二价锰离子.在与这些底物反应中,最适底物为过氧化氢(Km为3.7 tmmol/L).该酶具有抗过氧化氢漂白能力,锰过氧化物酶与高浓度的过氧化氢(2.5 mmol/L)作用60 min后仍能保持70％的活性.在所测试的染料中,锰过氧化物酶对结晶紫的脱色率最高达到65.8％.二价锰离子和过氧化氢对锰过氧化物酶脱色能力的影响进行研究,与孔雀绿相比,锰离子和过氧化氢对活性艳蓝脱色的影响很小.[结论]Trametes sp.SQ01锰过氧化物酶对过氧化氢的耐受性,以及对三苯甲烷类染料的高效脱色能力表明该酶在染料脱色降解方面有着广阔的应用前景.     

细菌脱色酶TpmD对三苯基甲烷类染料脱色的酶学特性研究

从嗜水气单胞菌DN322中分离纯化出能够对三苯基甲烷类染料结晶紫、碱性品红、灿烂绿及孔雀绿进行有效脱色的脱色酶,命名为TpmD。该酶的亚基分子量为29.4kDa,等电点为5.6。该酶催化上述4种三苯基甲烷类染料脱色反应的适合温度为40~60℃,适合pH范围为5.5~9.0。动力学参数测定结果显示TpmD对结晶紫、碱性品红、灿烂绿及孔雀绿的Km值分别为24.3、40.65、4.2、68.5μmol-1.L-1,Vmax值分别为19.6、74.1、82.8、115.6μmol.L-1.s-1。结晶紫为该酶的最适反应底物。TpmD催化的脱色反应依懒于NADH/NADPH及分子氧的存在,显示该酶属于NADH/NADPH依赖型的氧化酶类。这是国内外首次关于细菌中三苯基甲烷类染料脱色酶酶学性质的描述。     

白囊耙齿菌产锰过氧化物酶条件优化及其对染料的脱色

锰过氧化物酶（manganese peroxidase,MnP）是白腐真菌降解多种异生物质的主要降解酶之一。本研究对白囊耙齿菌Irpex lacteus产MnP的酶活曲线进行监测,利用单因素和正交试验对I. lacteus产MnP的发酵条件进行优化,同时检测了I. lacteus的MnP粗酶液对5种染料的脱色效果。结果显示,I. lacteus在培养5d时MnP活性较大;I. lacteus产MnP较优的条件为：可溶性淀粉20g/L、尿素1g/L、pH 6.3、CaCl₂ 1mmol/L、FeCl₃ 1mmol/L,该条件下MnP活性达29.24U/L,与优化前MnP活性相比提高了1.25倍;I. lacteus的MnP粗酶液对5种染料均可脱色,其中对直接大红和活性红的脱色效果更为明显,脱色5d后的脱色率分别达到82%和81%。     

烟管孔菌G14产锰过氧化物酶条件优化及其对染料的脱色

利用组织分离从未成熟有机蓝莓的表皮中分离出菌株G14,根据其菌落形态、ITS序列对比及系统发育树的分析,鉴定菌株G14为一株烟管孔菌Bjerkandera adusta.菌株G14可以分泌漆酶(laccase,Lac)、木质素过氧化物酶(lignin peroxidase,LiP)和锰过氧化物酶(manganese p...     

枸杞过氧化物酶的提取工艺优化及其酶学特性研究CSCD

枸杞为常用“药食同源”中药材,在加工、贮藏、运输等过程中易发生褐变,严重影响枸杞的外观和质量。前期研究发现枸杞中具有较高的过氧化物酶(peroxidase, POD)活性,而过氧化物酶在植物的酶促褐变中发挥着重要作用。为更好地控制枸杞酶促褐变的发生,本研究采用响应面法对枸杞过氧化物酶提取工艺进行优化,对枸杞过氧化物酶的性质进行了研究。结果显示枸杞过氧化物酶最佳提取工艺为料液比1∶3,浸提时间5 h,缓冲液pH为6.0,枸杞过氧化物酶活力为(5 148.59±50.00)U,与模型预测值接近,拟合性好,表明所得响应面模型可以很好地预测和分析枸杞过氧化物酶提取工艺条件。枸杞过氧化物酶最适温度为50℃,最适pH为6.0,金属离子Na~+、Ca~(2+)、K~+、Zn~(2+)、Cu~(2+)和柠檬酸对枸杞过氧化物酶起激活作用,抗坏血酸和亚硫酸能抑制枸杞过氧化物酶活性;该酶酶促反应动力学符合米氏方程,过氧化氢浓度一定时,酶对愈创木酚的K_m=21.52 mmol/L,V_(max)=115.47 U/mL;愈创木酚浓度一定时,酶对H_(2)O_(2)的K_m=1.41 mmol/L,V_(max)=148.81 U/mL。本研究可为后续枸杞加工过程中褐变的控制提供参考。     

The development of a thermostable CiP (Coprinus cinereus peroxidase) through in silico design

Protein thermostability is a crucial issue in the practical application of enzymes, such as inorganic synthesis and enzymatic polymerization of phenol derivatives. Much attention has been focused on the enhancement and numerous successes have been achieved through protein engineering methods. Despite fruitful results based on random mutagenesis, it was still necessary to develop a novel strategy that can reduce the time and effort involved in this process. In this study, a rapid and effective strategy is described for increasing the thermal stability of a protein. Instead of random mutagenesis, a rational strategy was adopted to theoretically stabilize the thermo labile residues of a protein using computational methods. Protein residues with high flexibility can be thermo labile due to their large range of movement. Here, residue B factor values were used to identify putatively thermo labile residues and the RosettaDesign program was applied to search for stable sequences. Coprinus cinereus (CiP) heme peroxidase was selected as a model protein for its importance in commercial applications, such as the polymerization of phenolic compounds. Eleven CiP residues with the highest B factor values were chosen as target mutation sites for thermostabilization, and then redesigned using RosettaDesign to identify sequences. Eight mutants based on the redesigns, were produced as functional enzymes and two of these (S323Y and E328D) showed increased thermal stability over the wild‐type in addition to conserved catalytic activity. Thus, this strategy can be used as a rapid and effective in silico design tool for obtaining thermostable proteins. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Phospholipid-enzyme interactions of chitin synthase of Coprinus cinereus

Abstract The effect of phospholipids on chitin synthase activity has been studied with digitonin-solubilized and partially purified preparations from Coprinus cinereus . When cholate was used as detergent, it inhibited enzyme activity, but this inhibition was reversed by increasing concentrations of phospholipids. Preincubation with cholate and phospholipid caused irreversible loss of activity. When sonicated with solubilized enzyme preparation, dimyristoyl phosphatidyl choline strongly stimulated activity, while dioleoyl phosphatidyl choline was inhibitory. The Arrhenius plot of the effect of temperature on enzyme activity contained breaks, characteristic of a membrane-bound enzyme. It is suggested that chitin synthase requires an annulus of phospholipids for activity.     

Removal of bisphenol A by polymerization and precipitation method using Coprinus cinereus peroxidase

Bisphenol A was efficiently removed by the polymerization and precipitation method using Coprinus cinereus peroxidase. The removal efficiency was optimal between pH 9–10 and at 40 °C with a molar ratio of H₂O₂ to bisphenol A of about 2. To remove 100 mg bisphenol A l^–1, peroxidase was required 5 U ml^–1 at pH 7 and 25 °C and 3 U ml^–1 at pH 10 and 40 °C.     

Kinetics of stem gravitropism in Coprinus cinereus: determination of presentation time and "dosage-response" relationships using clinostats

The sensitivity to gravitational stimulation of excised stems of the mushroom fruit body of Coprinus cinereus was determined using clinostat rotation to remove partially-stimulated stems from the normal unidirectional gravitational field. For the strain and conditions tested, the presentation time (the minimum time of stimulation required to elicit a gravitropic reaction) was determined to be 9.6 min. This is the first time the presentation time has been determined for a fungal gravitropic response. Constructional details are given of the clinostats employed in the research and their further use is discussed.     

Secretional expression of a Bacillus subtilis xylanase gene in the Basidiomycete Coprinus cinereus

The Bacillus subtilis endo (β-1,4-) D-xylanase structural gene (xyn) was trimmed away from its signal sequence and then fused after the signal sequence of the basidiomycete Pleurotus ostreatus manganese(II) peroxidase cDNA. The resulting modified gene (xyn′) was inserted between the promoter and terminator of two chromosome-integrating, heterologous protein expression vectors. These recombinant plasmids were introduced into protoplasts of the monokaryotic Coprinus cinereus trp1 strain with the C. cinereus TRP1-containing plasmid. One Trp⁺ Xyn⁺ transformant for each of the recombinant plasmids was obtained, which showed a markedly high xylan-degrading activity as compared with the control Trp⁺ transformant.     

Effect of temperature and pH on phenol removal using purified Coprinus cinereus peroxidase

The removal of phenol by peroxidase-catalysed polymerization was examined using purified Coprinus cinereus peroxidase. The phenol removal efficiency increased with a decrease in the reaction temperature over the range of 0–70 °C, though only a trace of enzyme activity with 4-aminoantipyrine (4-AAP), phenol and hydrogen peroxide was found at 0 °C. The optimum pH value for phenol removal was 9.0, while the enzyme expressed maximum activity at pH 7.5 in the presence of 4-AAP, phenol and hydrogen peroxide. By measuring residual enzyme activity in the polymerizing reaction mixture, it was shown that enzyme inactivation by free radicals was more suppressed at 0 °C than at 40 °C and that the adsorption of the enzyme on the polymerized precipitate was more suppressed at pH 9.0 than that at pH 7.5.     

Isolation of a LIM15/DMC1 homolog from the basidiomycete Coprinus cinereus and its expression in relation to meiotic chromosome pairing

The Escherichia coli gene recA is essential for homologous recombination and DNA repair, and homologs have been identified in eukaryotes. A basidiomycete, Coprinus cinereus, which has many advantages for the study of meiosis, was recently reported to have a homolog of one of these, RAD51. In the yeast Saccharomyces, mutations in the RAD5I gene cause defects in both somatic and meiotic cells. Based on this finding, we screened for a meiosis-specific homolog of recA, equivalent to Lilium LIM15 or Saccharomyces DMC1, in C. cinereus, and isolated a clone containing a 1.2-kb DNA fragment from a cDNA library constructed with Coprinus poly(A)⁺ RNA isolated from cells undergoing meiosis. The predicted amino acid sequence was 52% identical to the putative gene product of the lily cDNA clone LIM15 and 61% identical to Saccharomyces DMC1, and showed limited sequence similarity to the products of RAD52, 55, and 57. The synchrony of meiosis in Coprinus provides an ideal system for the investigation of differential gene expression in relation to meiosis and fruiting body development. Northern analysis indicated that Coprinus LIM15/DMC1 was expressed at meiotic prophase within 8 h after the onset of karyogamy, suggesting that the gene functions mostly at the stage at which the homologous chromosomes pair, but may not be essential at the point at which they recombine. The gene is not expressed in somatic cells. Received: 8 October 1998 / Accepted: 22 July 1999