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。     

灰盖鬼伞中过表达的碱性真菌漆酶在高盐染料废水中的应用潜力

【目的】 本研究利用双孢菇(Agaricus bisporus) gpdII启动子,实现了碱性真菌漆酶PIE5 (CcPIE5)在灰盖鬼伞(Coprinopsis cinerea) FA2222中的同源过表达。【方法】 在mKjalke液体培养基中37 ℃培养7 d,漆酶活力达到(24.2±1.1) U/mL。纯化的CcPIE5在pH 8.0和60 ℃下表现出最佳催化活性。【结果】 与已经研究报道的真菌漆酶不同,CcPIE5能耐受高浓度NaCl,当NaCl浓度从0升至1.5 mol/L时,CcPIE5的k_cat和K_m均呈下降趋势,表示CcPIE5在纺织印染废水脱色中有潜在应用价值。进一步在染料废水脱色应用中,以丁香酸作为介体,CcPIE5在pH 8.5和60 ℃条件下可高效降解(92.9±2.3)%的靛蓝胭脂红。通过LC-MS分析确认,Isatin 5-sulfonic acid (ISA)是靛蓝胭脂红降解的主要副产物。【结论】 CcPIE5在高温、碱性和含盐条件下高效脱色染料,是处理环境和工业特定应用的理想选择。     

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。     

枯草芽孢杆菌漆酶BsCotA的定向进化及其在靛蓝脱色中的应用

[目的]获得枯草芽孢杆菌(Bacillus subtilis)CotA漆酶活性及热稳定性提高的突变体并用于靛蓝脱色。[方法]利用易错PCR技术筛选获得2株酶活提高的漆酶突变体A1、A2,测定酶学性质,并进行靛蓝脱色研究。[结果]突变体A1、A2的最适pH值均为5.5,最适温度均为70℃;A1、A2在pH值2～7范围内,孵育30 min,剩余酶活均在40%以上,相比野生型(35%以下)有明显提高;A1、A2的比酶活为74.31 U/mg和80.96 U/mg,相比野生型(27.04 U/mg)分别提高了1.75倍、1.99倍;酶的三维结构分析显示,形成的4个突变位点中,1个突变位点通过氨基酸的替换,降低了主肽链的构象自由度,其余3个突变位点在漆酶的整体结构上引入了新的氢键;靛蓝染料脱色结果表明,3种漆酶对靛蓝的12 h脱色率均超过60%。[结论]筛选出来的漆酶突变体A1、A2,在pH值2～7范围内和65～80℃范围内的活性和稳定性比野生型显著提高,在靛蓝脱色等领域具有良好的应用基础。     

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。     

基于定向进化提高漆酶的催化活性及其在造纸制浆中的应用

在造纸工业中,漆酶作为一种生物催化剂,已经被广泛研究和应用。然而,天然漆酶在工业应用中存在着催化效率低、稳定性差等问题,限制了其在制浆工艺中的应用。为了进一步开发出酶活性高,耐受性强的漆酶,本研究对短小芽孢杆菌属来源漆酶进行定向进化改造,利用高通量筛选方法从随机突变体文库中筛选得到突变体F282L/F306L、Q275P,其比酶活分别为280.87 U/mg、453.94 U/mg,是野生型漆酶的1.42倍、2.30倍;突变体Q275P的温度稳定性有明显提升,其在40、50、70℃孵育4 h后的剩余酶活与野生型漆酶相比均提高了20%以上;突变体F282L/F306L和Q275P相较野生型漆酶对多种金属离子和有机溶剂的耐受性增强。野生型漆酶的K_m值为374.97 μmol/L,突变体F282L/F306L和Q275P的K_m值分别减小至318.96 μmo/L和360.71 μmo/L,底物亲和性有所提高;F282L/F306L和Q275P对底物2,2''-联氨-双(3-乙基苯并噻唑啉-6-磺酸)二胺盐[2,2''-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid),ABTS]的k_cat值分别为574.00 s^–1、898.03 s^–1,分别是野生型漆酶的1.1倍和1.7倍,催化效率较高。在处理纸浆应用中,突变体Q275P处理纸浆效果最好,相比于野生型漆酶,卡伯值降低了0.82,白度提高了2.00% ISO,抗张指数和裂断长分别提高7.8%和7.2%。本研究为漆酶更好地适应造纸工业环境奠定了基础。     

Highly enantioselective hydrolysis of dl-menthyl acetate to l-menthol by whole-cell lipase from Burkholderia cepacia ATCC 25416

Bisphenol A was polymerized by Coprinus cinereus peroxidase in aqueous 2-propanol solution. Various polymerized products with different molecular weights and hydroxyl values were synthesized depending on the reaction compositions (the ratio of aqueous buffer to 2-propanol). Poly(bisphenol A), a polymer of bisphenol A, was mixed with a diazonaphthoquinone derivative to form a new type of photoresist. A thin photoresist film was formed on the silicon wafer and exposed to UV light for different lengths of time. Poly(bisphenol A) having a molecular weight of approximately 3000 yielded sharply contrasted patterns as compared with the other poly(bisphenol A)s having different molecular weights.     

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.     

Peroxidase-benzhydroxamic acid complexes: spectroscopic evidence that a Fe-H2O distance of 2.6 Å can correspond to hexa-coordinate high-spin heme

Resonance Raman (RR) spectra have been obtained for single-crystal horseradish peroxidase isozyme C complexed with benzhydroxamic acid (BHA). The data are compared with those obtained in solution by both RR and electronic absorption spectroscopies at room and low (12-80 K) temperatures. Moreover, the analysis has been extended to Coprinus cinereus peroxidase complexed with BHA. The results obtained for the two complexes are very similar and are consistent with the presence of an aqua six-coordinate high-spin heme. Therefore it can be concluded that despite the rather long Fe-H2O distance of 2.6-2.7 A found by X-ray crystallography in both complexes, the distal water molecule can still coordinate to the heme iron.     

改进酶稳定性的定向进化技术

酶作为一种生物催化剂,以其独特的优良特性,在绿色化学和清洁生产中得到了广泛的应用。随着酶定向进化技术的建立和发展,通过定向进化改进酶稳定性的研究越来越多。详细综述了各种定向进化方法的特点及在提高酶稳定性方面的应用,并从结构和功能的角度进一步解释了相关机理。     

A distinctive phenotype in the common-<Emphasis Type="Italic">A</Emphasis> heterokaryon of <Emphasis Type="Italic">Coprinus cinereus</Emphasis>

The homobasidiomycete Coprinus cinereus, unlike Schizophyllum commune, is not known to exhibit an obvious heterokaryotic phenotype in common-A matings. In the present study we found that progeny isolated from a fruit-body collected in the field exhibit a distinctive mycelial development in common-A matings. Genetic analysis suggested that the common-A heterokaryotic phenotype is brought about by a nuclear factor(s) other than the mating type genes. Received: March 30, 2001 / Accepted: October 1, 2001     

微生物酶分子改造研究进展

近年来,越来越多的酶蛋白已经采用重组微生物反应器进行高效生产。为了改善酶蛋白的催化性能,提高其环境适应性,同时提高酶蛋白的表达量,降低生产成本,各种针对酶蛋白分子改造的基因工程技术已经得到大量的应用。综述了用于酶分子改造和进化的主要分子生物学方法,如定点突变、易错PCR、基因改组、密码子优化等技术及其应用成就。