烟管孔菌G11对活性染料的脱色

本研究从未成熟的有机蓝莓表皮分离、纯化得到一株白腐真菌G11,通过对菌株G11的形态特征、ITS序列同源性比对以及系统发育分析,鉴定菌株G11为一株烟管孔菌Bjerkandera adusta。菌株G11可以产生木质素过氧化物酶、漆酶和锰过氧化物酶等木质素降解酶。菌株G11对8种不同染料的脱色效果显示其对活性染料的脱色效果最好,脱色率达到90%所需时间最短。以菌株G11为研究对象,研究其对不同浓度的活性黑和活性红的脱色能力,结果表明：菌株G11对活性红和活性黑具有显著的脱色能力。在脱色15d时,菌株G11对浓度为10、50、100、250、500mg/L活性红的脱色率分别为99%、98%、95%、94%和92%;对浓度为10、50、100、250、500mg/L活性黑的脱色率分别为98%、97%、95%、93%和90%。     

酵母绿色荧光蛋白报告载体的构建及其在过氧化物酶体研究中的应用

以载体pYES2为基础,构建了酵母表达载体pYES2G,该载体含有融合了过氧化物酶体定位信号1(PTS1)的绿色荧光蛋白报告分子GFP-SKL编码基因,该基因以酵母TEF1启动子启动。pYES2转化研究表明,在野生型酵母INVScl中,GFP-SKL蛋白在细胞中呈点状聚集,而在酵母PEX5p缺陷菌株ATCC4003603中,荧光为弥散状,证明报告分子GFP-SKL可通过PEX5p蛋白有效定位到过氧化物酶体。在载体pYES2G的多克隆位点分别连入酵母及产黄青霉PEX5p编码基因得到载体pYES2G/ScPEX5和pYES2G/PcPEX5,转化酵母ATCC4003603,荧光均呈聚集状,证明外源PEX5p基因的表达恢复了缺陷菌株的功能。pYES2G载体为真菌过氧化物酶体相关基因的功能研究提供了直观有效的方法。     

裂褶菌菌株G18对孔雀石绿染料的脱色优化

通过对兔眼蓝莓幼果组织中分离得到的内生真菌G18进行形态特征、ITS序列和系统进化分析鉴定菌株G18为裂褶菌Schizophyllum commune。同时,对菌株G18产生的3种木质素降解酶进行监测,发现G18菌株可以分泌漆酶、木质素过氧化物酶和锰过氧化物酶。为明确裂褶菌G18对染料的脱色能力,利用裂褶菌G18对固体条件下8种染料进行脱色能力的检测,筛选出较易脱色的染料后,对该染料的脱色条件进行优化。结果表明,裂褶菌G18对8种染料均可以脱色,对孔雀石绿染料的脱色效果最好。裂褶菌G18对孔雀石绿的脱色优化结果为pH 7.0、20.0g/L淀粉、1.0g/L尿素、1.0g/L硫酸锌、接菌量9片（d=5.0mm）。     

链格孢柑橘致病型过氧化物酶体对接复合体蛋白AaPex13和AaPex14的生物学功能

过氧化物酶体是存在于真核细胞中的一类单层膜细胞器,参与多种生理生化代谢过程,而Pex13和Pex14是过氧化物酶体膜上的对接复合体蛋白,参与基质蛋白-受体复合体的跨膜运输。目前,Pex13和Pex14在大多数植物病原真菌中的生物学功能尚不清楚。本研究鉴定了柑橘褐斑病菌链格孢柑橘致病型(the tangerine pathotype of Alternaria alternata)的对接复合体蛋白Pex13和Pex14,并构建基因敲除突变体与回补菌株,探究其生物学功能。结果表明,与野生型和回补菌株相比,ΔAaPex13和ΔAaPex14营养生长、分生孢子形成显著下降,分生孢子的萌发率显著降低,抗氧化能力和抗细胞壁胁迫能力也显著减弱,病菌的ACT毒素产量分别降低30%和33%,在离体叶片上丧失致病力。此外,AaPex13和AaPex14的缺失导致基质蛋白无法定位到过氧化物酶体,过氧化物酶体生物发生存在缺陷。本研究明确了AaPex13和AaPex14在病菌生长发育、过氧化物酶体形成、ACT毒素产生以及维持致病力方面都具有重要的调控作用。     

甘草药渣降解菌的筛选及其产酶工艺研究

筛选能够有效降解甘草药渣的菌株,优化菌株的纤维素酶生产工艺。从腐烂的甘草及土壤中筛选甘草药渣降解菌,结合形态学观察及18S r DNA测序确定菌株的分类地位;考察了不同因素对菌株发酵的影响;采用L9(34)正交分析法确定菌株最佳产酶条件;利用菌株生产的纤维素酶对甘草药渣进行酶解研究。确定分离菌株为Penicillium oxalicum,命名为草酸青霉G2。正交分析结果表明,药渣含量、发酵时间对G2的发酵有显著影响(P0.05,P0.05);在最佳产酶工艺条件下,测得G2滤纸酶活力3.43 U/m L、内切酶活力16.89 U/m Lu;甘草药渣的酶解结果表明,G2生产的纤维素酶(G2酶)对甘草药渣的酶解效率优于商品酶。此外,甘草药渣经酶解以后,甘草总黄酮提取率明显升高。对甘草药渣降解菌的筛选以及对菌株的产酶工艺研究,旨在为合理利用甘草药渣以及生产成本低廉、性能优良的纤维素酶奠定基础。     

同工酶研究在嗜热脂肪芽孢杆菌分类中的应用

嗜热脂肪芽孢杆菌是一个尚未被严格定义的种,它几乎包括芽孢杆菌属内所有能在65℃以上生长的细菌。用聚丙烯酰胺凝胶电泳的方法对151株嗜热脂肪芽孢杆菌的9种酶(G6PDH、LDH、MDH、IDH、AlaDH、LeuDH、过氧化氢酶、过氧化物酶、酯酶)的酶谱进行测定分析。根据其酶谱的差异,可将151株菌株分成两个类型。两型之间8个酶的13或14个基因产物的相异度约为91.8％,两型间的标准遗传距离是2.55。因此这两种类型的细菌也许可以被看作为两个不同的种。     

毕赤酵母G5拮抗葡萄灰霉病机理初探

针对新疆红提葡萄中分离的生防菌株——毕赤酵母G5,以葡萄灰霉菌(Botrytis cinerea)为靶标菌,研究了G5对葡萄灰霉菌孢子及对葡萄果实内5种关键酶活性的影响,初步探讨了G5拮抗葡萄采后灰霉病的机理。采用离体(in vitro)实验与活体(in vivo)实验的方法对毕赤酵母菌G5拮抗葡萄灰霉病的机理进行探究。结果发现,毕赤酵母菌株G5对葡萄灰霉菌孢子的萌发和菌丝生长均有抑制作用,对灰霉菌丝的抑制率最高为80.20%,对灰霉孢子抑制率达86.89%;两者间存在营养竞争关系,有重寄生现象产生,拮抗菌自身并不分泌抗菌物质。此外,该菌株能诱导果实体内过氧化物酶、多酚氧化酶、苯丙氨酸解氨酶、几丁质酶、β-1,3-葡聚糖酶的酶活性,使其酶活性有显著提高,说明毕赤酵母G5可以有效的诱导果实内抗病相关酶的活性,增强对灰霉病的抑制效果。毕赤酵母G5拮抗葡萄灰霉病的机理主要包括营养竞争、诱导抗病性,是否有重寄生作用还需要进一步验证。     

黄孢原毛平革菌抗营养阻遏产漆酶诱变育种及其产酶特性

【目的】筛选能抗营养阻遏产漆酶的黄孢原毛平革菌,论证其产漆酶的确定性及抗营养阻遏产木质素酶的可行性,为白腐菌产酶代谢调控、木质素降解机理的研究奠定基础。【方法】利用重复紫外诱变法,以愈创木酚富氮鉴别培养基筛选目标菌株;比较不同营养条件下菌体生长与产酶动力学差异研究产酶营养调控机理;通过热处理、排除锰离子和加入过氧化氢酶等不同措施论证黄孢原平毛平革菌能否产生漆酶。【结果】3种不同方法均证实选育到的pcR5305和pcR5324菌株在限氮与富氮条件下均能产生漆酶,pcR5305和pcR5324在限氮条件下产漆酶分别达到203.5、187.6 U/L;在富氮条件下为220.6、183.9 U/L,而原菌株pc530在两种条件下都基本不产生漆酶。二菌株产漆酶调控方式不同,pcR5305漆酶产生与菌体生长同步,而pcR5324漆酶产生却受营养氮阻遏。二菌株同时具有抗营养阻遏高产木质素过氧化物酶(LiP)和锰过氧化物酶(MnP)(分别为LiP 1343.2、MnP 252.2 U/L;LiP 1169.5、MnP 172.4 U/L)的能力。【结论】筛选到的黄孢原毛平革菌变异菌株能产漆酶,同时表现了抗营养阻遏产漆酶、木质素过氧化物酶和锰过氧化物酶的能力,具有重要的生产应用与理论研究价值,为白腐菌产酶代谢调控机理研究提供了原始菌株并奠定了良好的基础。     

酿酒酵母Mbp1基因缺失突变株耐受性研究

为了研究Mbp1基因对工业酿酒酵母(Saccharomyces cerevisiae)耐受性的影响,在敲除野生型工业酿酒酵母MF1015菌株Mbp1基因的基础上,分析Mbp1基因缺失菌株(突变菌株)与野生型在乙醇耐受性、耐热性、细胞壁完整性、呼吸强度、海藻糖含量、过氧化氢酶(CAT)、过氧化物酶(POD)、超氧化物歧化酶(SOD)、乙醇脱氢酶(ADH)活性等方面的差异。结果表明:在含刚果红或SDS的平板上突变菌株的菌落比野生型小、菌体存活率比野生型低;突变菌株的乙醇耐受性和耐热性均下降,在乙醇浓度为9%的液体培养基中,突变菌株的最大OD600约为3.15,而野生型最大OD600值约为3.48;在30℃和37℃时突变菌株和野生型的生长趋势基本一致,而在40℃时,突变菌株最大OD600约为3.0,而野生型约为4.5;用9%乙醇处理后,突变菌株胞内海藻糖含量、过氧化氢酶、过氧化物酶、超氧化物歧化酶、乙醇脱氢酶酶活分别比野生型低49%、80%、24%、37%、73%,而未经乙醇处理的突变菌株和野生型酶活无明显差别。推测Mbp1基因有助于工业酿酒酵母适应外部不良环境。     

香蕉乙二醛酶基因增强酿酒酵母对非生物胁迫抵抗能力的研究

从香蕉cDNA文库中克隆到了一个香蕉(Musa acuminata AAA subgroup)乙二醛酶(glyoxalase,GLO)基因(MaGLO14)。构建了带有MaGLO14的酵母表达载体PYES2-MaGLO14,转化酿酒酵母(Saccharomyces cerevisiae)尿嘧啶营养缺陷型菌株INVSC1,挑取转化子进行PCR和酶切鉴定,证实获得了转基因菌株。通过比较转基因菌株和非转基因菌株在NaCl、高温、低温、干旱、UV胁迫下的生长状况,证明转基因菌株在以上非生物胁迫条件下的存活菌落数均高于非转基因菌株。利用酿酒酵母初步证明MaGLO14具有增强酵母菌对非生物胁迫抵抗力的功能。     

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%。     

Rapid decolorization of azo dyes by crude manganese peroxidase from Schizophyllum sp. F17 in solid-state fermentation

The production of ligninolytic enzymes by the fungus Schizophyllum sp. F17 using a cost-effective medium comprised of agro-industrial residues in solid-state fermentation (SSF) was optimized. The maximum activities of the enzymes manganese peroxidase (MnP), laccase (Lac), and lignin peroxidases (LiP) were 1,200, 586, and 109 U/L, respectively, on day 5 of SSF. In vitro decolorization of three structurally different azo dyes by the extracellular enzymes was monitored to determine its decolorization capability. The results indicated that crude MnP, but not LiP and Lac, played a crucial role in the decolorization of azo dyes. After optimization of the dye decolorization system with crude MnP, the decolorization rates of Orange IV and Orange G, at an initial dye concentration of 50 mg/L, were enhanced to 76 and 57%, respectively, after 20 min of reaction at pH 4 and 35°C. However, only 8% decolorization of Congo red was observed. This enzymatic reaction system revealed a rapid decolorization of azo dyes with a low MnP activity of 24 U/L. Thus, this study could be the basis for the production and application of MnP on a larger scale using a low-cost substrate.     

Decolorization of textile dyes by whole cultures of Ischnoderma resinosum and by purified laccase and Mn-peroxidase

Ischnoderma resinosum produced extracellular ligninolytic enzymes laccase and MnP. The activity of laccase achieved the maximum on day 10 (29.4 U L⁻¹), the MnP on day 14 (34.5 U L⁻¹). Laccase and Mn-peroxidase were purified from the culture liquid using gel permeation and ion-exchange chromatographies. Purified Mn-peroxidase performed decolorization of all textile dyes tested (Reactive Black 5, Reactive Blue 19, Reactive Red 22 and Reactive Yellow 15). Laccase was inactive with Reactive Black 5 and Reactive Red 22, while all dyes were decolorized after addition of the redox mediators violuric acid (VA) and hydroxybenzotriazole (HBT). The culture liquid from I. resinosum cultures was also able to decolorize all dyes as well as the synthetic dyebaths in the presence of VA and HBT. The highest decolorization rates were detected in acidic pH (3–4).     

三种白腐真菌脱色噻嗪染料亚甲基蓝

本研究通过含亚甲基蓝染料的固体培养基,从19株白腐真菌菌株中分离获得3个脱色能力较强的菌株,其在平板上的脱色圈大小分别为7.5cm、6.8cm和5.5cm。鉴定其为：云芝栓孔菌Trametes versicolor（ZT-197）,绒毛栓孔菌Trametes pubescens（ZT-230）和亚黑管孔菌Bjerkandera fumosa（ZT-307）。其中,ZT-230对染料亚甲基蓝的脱色能力最强,可以将染料浓度为50mg/L的100mL液体培养基在6d之内100%脱色,而ZT-197和ZT-307在接种第10天时的脱色率为98%和80%。同时测定了3株白腐真菌在降解染料过程中的漆酶、锰过氧化物酶和木素过氧化物酶3种酶活力的规律：ZT-197和ZT-230均可分泌Lac和MnP两种酶,ZT-307只分泌LiP。本研究说明绒毛栓孔菌ZT-197在印染废水治理方面具有较好的应用前景。     

高效木质素降解菌的筛选及其对玉米秸秆的降解效果

本研究利用愈创木酚和苯胺蓝固体培养基对菌株进行初筛,利用形态学和分子生物学对筛选出的菌株进行鉴定,以黄孢原毛平革菌Phanerochaete chrysosporium CGMCC 5.0776为对照,利用其对玉米秸秆进行预处理并测定木质素和纤维素的降解率,测定筛选菌株在预处理玉米秸秆过程中漆酶、锰过氧化物酶（manganese peroxidase,MnP）和木质素过氧化物酶（lignin peroxidase,LiP）活性。结果表明:利用愈创木酚和苯胺蓝固体培养基,从16株白腐真菌菌株中筛选出2株具有较高漆酶或MnP活性的菌株,鉴定其为桦栓孔菌Trametes betulina (L.) Pilát（ZT-153）和亚黑管孔菌Bjerkandera fumosa (Pers.) P. Karst.（ZT-307）,测定T. betulina ZT-153和B. fumosa ZT-307对玉米秸秆酸不溶木质素降解效率分别为13.60%和21.87%,较对照P. chrysosporium CGMCC 5.0776高1.58%和9.85%,对纤维素的降解率较低,分别为4.10%和4.50%。2株菌株在预处理玉米秸秆过程中,T. betulina ZT-153表现出漆酶和MnP活性,B. fumosa ZT-307只表现出LiP活性。其中B. fumosa ZT-307对玉米秸秆酸不溶木质素的降解效率最高,在秸秆资源的综合利用方面具有较好的潜力和应用前景。     

Reaction of manganese-dependent peroxidase from Bjerkandera adusta in aqueous organic media

The oxidation of various phenolics and aromatic amines by manganese-dependent peroxidase (MnP) of Bjerkandera adusta was examined in aqueous organic media. MnP retained its activities in several 70% (v/v) aqueous solutions of water-miscible organic solvents including ethylene glycol, diethylene glycol, acetone and acetonitrile. The absorption spectra of MnP in these aqueous organic media were similar to that observed in the reaction without solvent addition, indicating that the heme of MnP was little affected by the addition of these water-miscible organic solvents. MnP was also found to oxidize Mn(II) to Mn(III) in these 70% (v/v) aqueous organic media. The oxidation of Mn(II) by MnP was correlated with the Dimroth–Reichardt parameter, E_T(30), of the solvents. Furthermore, MnP catalyzed the oxidation of anisidines, aminophenols, phenylenediamines and phenolics in aqueous 70% (v/v) acetone, acetonitrile and diethylene glycol media. Aromatic amines that have high hydrophobicity were shown to be suitable for the reaction of MnP in aqueous water-miscible organic media.     

液态发酵条件下拟康宁木霉8985产纤维素酶能力初探

液态发酵条件下,以微晶纤维素为唯一碳源,比较了拟康宁木霉Trichoderma koningiopsis 8985和里氏木霉T. reesei QM9414产纤维素酶的能力。8985发酵12 h开始产生纤维素酶,36 h时酶活达到产酶峰值的50%,此时QM9414尚未诱导产酶。测定8985发酵84 h时上清液中滤纸纤维素酶、羧甲基纤维素酶、β-葡萄糖苷酶和木聚糖酶的酶活分别为1.06、3.62、1.80和6.67 IU/mL,分别是QM9414上述酶活的1.72、1.70、6.35和1.12倍。8985滤纸纤维素酶酶活的最适反应条件为pH 4.5,反应温度50 ℃,在Fe³⁺ (≤ 4 mmol/L)和Cu²⁺ (0-10 mmol/L)存在条件下酶活稳定。     

Decolorization of Victoria blue by the white rot fungus, <Emphasis Type="Italic">Phanerochaete chrysosporium</Emphasis>

Synthetic textile dyes are among the most dangerous chemical pollutants released in industrial wastewater streams. Recognizing the importance of reducing the environmental impact of these dyes, the ability of the white rot fungus Phanerochaete chrysosporium to decolorize various textile dyes was investigated. This fungus decolorized 6 of the 14 structurally diverse dyes with varying efficiency (between 14% and 52%). There was no discernable pattern of decolorization even among dyes of the same chemical class, suggesting that attack on the dyes is relatively non-specific. Among the three dyes which showed >40% decolorization, Victoria Blue B (VB) was chosen for further analysis because the ability of the fungus to decolorize VB was nearly independent over a relatively broad concentration range. Blocking lignin peroxidase (LiP) and manganese peroxidase (MnP) production by the fungus did not substantially affect VB decolorization. Inhibition of laccase production by adding various inhibitors to shaken cultures reduced VB decolorization significantly suggesting a role for laccase in VB decolorization. When sodium azide and aminotriazole were used to inhibit endogenous catalase and cytochrome P-450 oxygenase activities, there was 100% and 70% reduction in VB decolorization, respectively. Adding benzoate to trap hydrogen peroxide-derived hydroxyl radicals resulted in 50% decolorization of VB. Boiling the extracellular fluid (ECF) for 30 min resulted in approximately 50% reduction in VB decolorization. Collectively, these data suggest that laccase, and/or oxygenase/oxidase and a heat-stable non-enzymatic factor, but not Lip and MnP, play a role in VB decolorization by P. chrysosporium.