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 peroxidase,MnP）3种木质素降解酶,利用单因素和正交试验对活性较高的MnP进行发酵条件优化,同时检测B.adustaG14所产MnP粗酶液对5种染料的脱色能力。结果表明,B.adustaG14在培养6d时MnP活性最大,最优条件为：蔗糖10g/L、pH 7、0.5mmol/L Mn²⁺、0.1mmol/L Zn²⁺,该条件下MnP活性达17.74U/L,比优化前提高了1.42倍,B.adustaG14 MnP粗酶液对5种染料均可以脱色,对刚果红和铬黑T染料的脱色效果最好,6d后脱色率达76%和68%。     

裂褶菌F17对刚果红的脱色及主要降解酶的研究

裂褶菌F17在限氮培养基中对偶氮染料刚果红表现出较高的脱色能力。最佳脱色条件为:温度28℃、初始pH值4.5以及菌体摇瓶培养3d加入染料,染料浓度以100mg/L为宜。诱导因子藜芦醇、吐温80、土豆汁和松木屑的加入明显提高了脱色率,而叠氮化钠和氰化钾的加入对脱色有显著的抑制作用。酶活检测表明,裂褶菌F17在刚果红脱色过程中主要产生MnP,LiP活力很小,未检测到Lac。此外,刚果红的脱色率与累积MnP酶活具有良好的线性关系,相关系数为0.973。推测裂褶菌F17对刚果红脱色的主要降解酶为MnP。     

Ganoderma lucidum U-281漆酶催化偶氮染料活性黑5脱色

漆酶在纺织染料脱色及印染废水处理领域有着广阔的应用前景。活性黑5是纺织印染中应用广泛的偶氮类活性染料,结构复杂,生物降解性低。以灵芝菌Ganoderma lucidum U-281所产漆酶对活性黑5进行氧化脱色,采用单因素逐一优化方法得到了U-281漆酶催化活性黑5脱色的工艺参数：染料初始浓度25mg/L、漆酶用量2.0U/mL、铜离子添加量40mmol/L、pH 6.0、40℃。在优化条件下,4h可使RB5脱色62.34%,24h可完成90%以上的脱色效果。     

不同培养基对富集筛选脱色真菌菌群的效果比较

利用活性黑RB5和活性红M-3BE作为筛选因子,从染料脱色效果、菌群产酶能力以及菌群中的微生物丰富度三方面比较了酵母培养基A、产漆酶真菌培养基B和白腐真菌培养基D在脱色真菌富集筛选方面的效果。富集筛选结果共得到11组具有明显脱色效果的真菌菌群,其中5组来自于D培养基,A和B培养基各获得3组。来自A培养基的3组菌群显示出最好的脱色效果和最大的菌群丰富度,对50mg/L的活性红M-3BE和酸性红A溶液的脱色率最高达到99.53%和97.42%,从中分离到了16株真菌,初步鉴定分属于水霉科、曲霉科(红曲霉属)、节壶菌科和白粉菌科;而B和D培养基中所获得的菌群脱色效果稍差,从中仅得到3株和2株真菌,初步鉴定属于酵母和青霉。A、B两种培养基在各种染料存在下更易产生木质素过氧化物酶,产漆酶能力较弱,而D培养基产漆酶活性较高。     

裂褶菌F17锰过氧化物酶酶活力影响因素的响应面优化(英文)

锰过氧化物酶是真菌分泌的一种糖基化的含有血红素辅基的胞外蛋白,在染料降解和脱色过程中起着重要作用。本实验利用本实验室保存的的白腐真菌裂褶菌Schizophyllum sp.F17产锰过氧化物酶(MnP),研究MnP的酶学性质,并对酶活条件进行优化。实验通过超滤浓缩、DEAE-纤维素、DE52离子交换层析和Sephadex G-75凝胶过滤等步骤,分离纯化得到电泳纯的锰过氧化物酶。该酶蛋白含量为23μg/mL,分子量大小为49.2kDa,在0.1mmol/L H2O2中半衰期为5~6min。Mn2+、H2O2以及酶的用量可以影响MnP酶促反应的效率,在单因子分析法的基础上,通过全因子中心组合设计响应面分析表明:H2O2以及H2O2与酶用量之间的交互作用对酶促反应的作用是最显著的。在优化条件下,酶对偶氮染料金橙G、刚果红显示出较强的脱色能力。     

短小芽孢杆菌LC01漆酶基因在毕赤酵母中的胞外表达及重组漆酶酶学性质研究

为了获得表达量高、稳定性好及染料脱色效率高的细菌漆酶,通过PCR扩增出短小芽孢杆菌LC01的漆酶基因并构建重组表达载体pPICZαA-lac,转化毕赤酵母菌株SMD1168H后利用甲醇诱导培养重组菌获得重组漆酶,纯化并分析了重组漆酶的性质。重组菌株产漆酶活性在第7天达到最高,为1 390 U/L。纯化的重组漆酶分子量为65 kD,以丁香醛连氮为底物的最适反应温度和pH分别为70℃和6.8。在pH 9.0放置10 d活性没有下降,在70℃保温10 h后仍保留36%的酶活。Al~(3+)、Fe~(3+)和Mn~(2+)完全抑制漆酶活性。在介体乙酰丁香酮参与下该漆酶能够有效脱色RB亮蓝、活性黑5和靛红,在pH 9.0时6 h的脱色率达到了90%以上,表明该重组漆酶能有效应用于染料废水的脱色处理。     

阿魏菇与胶红酵母共培养产漆酶对活性艳蓝W-RV的脱色

利用阿魏菇与胶红酵母共培养所产漆酶对染料活性艳蓝W-RV进行脱色,同时考察不同p H、温度、染料浓度和漆酶酶活等条件对脱色的影响。结果显示,酸性范围内的p H有利于活性艳蓝W-RV的脱色,较高温度并不适于脱色反应。在研究不同染料浓度的影响时发现,不同浓度染料的脱色率在7 h反应后都达到了稳定,而漆酶酶活在达到200 U/L后脱色率不再变化。因此,最终得到的最适反应条件为p H 4.5、30℃、染料质量浓度100 mg/L、漆酶酶活200 U/L,在此条件下,活性艳蓝W-RV的最高脱色率为89.91%。     

不同培养基对富集筛选脱色真菌菌群的效果比较

利用活性黑RB5和活性红M-3BE作为筛选因子,从染料脱色效果、菌群产酶能力以及菌群中的微生物丰富度三方面比较了酵母培养基A、产漆酶真菌培养基B和白腐真菌培养基D在脱色真菌富集筛选方面的效果。富集筛选结果共得到11组具有明显脱色效果的真菌菌群,其中5组来自于D培养基,A和B培养基各获得3组。来自A培养基的3组菌群显示出最好的脱色效果和最大的菌群丰富度,对50mg/L的活性红M-3BE和酸性红A溶液的脱色率最高达到99.53%和97.42%,从中分离到了16株真菌,初步鉴定分属于水霉科、曲霉科（红曲霉属）、节壶菌科和白粉菌科;而B和D培养基中所获得的菌群脱色效果稍差,从中仅得到3株和2株真菌,初步鉴定属于酵母和青霉。A、B两种培养基在各种染料存在下更易产生木质素过氧化物酶,产漆酶能力较弱,而D培养基产漆酶活性较高。     

一株产漆酶真菌新月弯孢霉JQH-100在染料脱色中的应用

从感染叶斑病的玉米叶片中分离、纯化得到一株高产漆酶的新月弯孢霉Curvularia lunata JQH-100菌株。液体培养Curvularia lunata JQH-100可产漆酶且活性较高,产酶高峰出现在第3天;以ABTS为底物粗酶液的最适反应温度是30℃,最适反应pH是2.8;染料脱色的研究表明,共培养体系对茜素红的脱色率达到了92.6%,对中性红和刚果红的脱色率也都在80%以上;Curvularia lunata JQH-100所产漆酶经纯化后对染料茜素红和刚果红有较高的脱色率,分别为82.1%和81.2%。研究结果显示Curvularia lunata JQH-100在染料废水处理中有较大应用潜力。     

Decolorization of sulfonphthalein dyes by manganese peroxidase activity of the white-rot fungus<Emphasis Type="Italic">Phanerochaete chrysosporium</Emphasis>

Manganese peroxidase (MnP) was produced by shallow stationary cultures of Phanerochaete chrysosporium growing on N-limited medium. Decolorization of sulfonphthalein (SP) dyes by MnP was investigated. The MnP activity profile and decolorization of SP dyes was correlated and almost all dyes were decolorized at pH 4.0. The influence of various inhibitors on Bromocresol Purple decolorization suggested an oxidative nature of the MnP-catalyzed decolorization of SP dyes.     

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.     

Roles of Lignin Peroxidase and Manganese Peroxidase from Phanerochaete chrysosporium in the Decolorization of Olive Mill Wastewaters

The relative contributions of lignin peroxidase (LiP) and manganese peroxidase (MnP) to the decolorization of olive mill wastewaters (OMW) by Phanerochaete chrysosporium were investigated. A relatively low level (25%) of OMW decolorization was found with P. chrysosporium which was grown in a medium with a high Mn(II) concentration and in which a high level of MnP (0.65 (mu)M) was produced. In contrast, a high degree of OMW decolorization (more than 70%) was observed with P. chrysosporium which was grown in a medium with a low Mn(II) concentration but which resulted in a high level of LiP activity (0.3 (mu)M). In this culture medium, increasing the Mn(II) concentration resulted in decreased levels of OMW decolorization and LiP activity. Decolorization by reconstituted cultures of P. chrysosporium was found to be more enhanced by the addition of isolated LiP than by the addition of isolated MnP. The highest OMW decolorization levels were obtained at low initial chemical oxygen demands combined with high levels of extracellular LiP. These data, plus the positive effect of veratryl alcohol on OMW decolorization and LiP activity, indicate that culture conditions which yield high levels of LiP activity lead to high levels of OMW decolorization.     

Decolorization of the azo dye Acid Red 18 by crude manganese peroxidase: Effect of system parameters and kinetic study

Abstract

To optimize operating conditions for the decolorization of the azo dye Acid Red 18 (AR18) by crude manganese peroxidase (MnP), some important factors affecting enzymatic decolorization were systematically investigated. Under the optimal enzyme reaction conditions, a decolorization efficiency of more than 82.3% was achieved after 60 min treatment. Furthermore, the manganese chelators, malate, tartrate, and lactate were found to be more favorable for the decolorization of AR18 than malonate, acetate, succinate, maleate, oxalate, and citrate. However, the presence of NaCl or Na₂SO₄ had a negative impact on the decolorization of AR18. The K_m and V_max values of MnP for AR18 were 169.66 μmol L^{? 1} and 20.63 μmol L^{? 1} min^{? 1}, respectively. The decolorization of AR18 by MnP followed second-order reaction kinetics with respect to the dye concentration. The decolorization rate constant increased with increasing temperature from 20°C to 35°C, which indicated an activation energy (E_a) of 15.87 kcal mol^{? 1} and frequency factor (k₀) of 1.36 × 10⁸ mg^{? 1} L min^{? 1} according to the Arrhenius equation. The results obtained provide experimental data for the application of crude MnP for the decolorization of AR18, and help to elucidate the biochemical mechanism of dye decolorization by the enzyme.     

Production of manganese-dependent peroxidase in a new solid-state bioreactor by Phanerochaete chrysosporium grown on wood shavings. Application to the decolorization of synthetic dyes

The production of manganese-dependent peroxidase (MnP) byPhanerochœte chrysosporium in a new solid-state bioreactor, the immersion bioreactor, operating with lignocellulosic waste, such as wood shavings, was investigated. Maximum MnP and lignin peroxidase (LiP) activity of 13.4 and 8.48 μkat/L were obtained, respectively. Thein vitro decolorization of several synthetic dyes by the extracellular liquid produced in the above-mentioned bioreactor (containing mainly MnP) was carried out and its degrading ability was assessed. The highest decolorization was reached with Indigo Carmine (98%) followed by Bromophenol Blue (56%) and Methyl Orange (36%), whereas Gentian Violet was hardly decolorized (6%).     

Decolorizing an anthraquinone dye by Phlebia brevispora: Intra‐species characterization

Remazol brilliant blue R (RBBR) is an anthraquinone dye derived from anthracene that is decolorized by a white rot fungus, Phlebia brevispora. Interestingly, P. brevispora produces two phenomena of yellowish and pinkish colors during the degradation of RBBR. Here, we characterized the decolorization of RBBR by P. brevispora. The fungus was significantly different between the two colors via UV spectrophotometry, and the morphology of the hyphae observed in the respective color culture was also entirely different. Moreover, both of the two ligninolytic enzymes, laccase and manganese‐dependent peroxidase (MnP), were remarkably stimulated in the yellowish culture at the beginning of the decolorization. It is possible that the RBBR decolorizing mechanism might be primarily related to the amount of laccase and MnP produced in the yellowish culture. Thus, the decolorized color may be rapidly estimated at initial period of incubation. In addition, GeneFishing technology revealed that two genes were differentially expressed in yellowish culture.     

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.     

Improvements in the determination of manganese peroxidase activity

The existing method of determining the activity of manganese peroxidase (MnP), produced by Phanerochaete chrysosporium, was improved. 2,6-Dimethoxyphenol at 80 mM was used as a substrate and, after the decolorization of the reaction mixture, H₂O₂ was added and the initial reaction rate was used to determine MnP activity.     

In vitro degradation of a polymeric dye (Poly R-478) by manganese peroxidase

The aim of this study is the evaluation of the enzymatic action of the ligninolytic enzyme manganese peroxidase (MnP), through a suitable addition of H(2)O(2), as a feasible system for the in vitro degradation of complex structures. For this purpose, a highly recalcitrant polymeric dye (Poly R-478) was selected as a model compound. An amperometric technique was used to determine the H(2)O(2) requirement in the decolorization by nonpurified MnP. Two H(2)O(2) supply strategies-fed-batch (every hour) or semicontinuous (every 5 min)-were applied. The addition of H(2)O(2) in pulses led to a limited decolorization after the pulses and the instantaneous consumption or decomposition of H(2)O(2). Therefore, this way of addition may limit the actual H(2)O(2) concentration in the reaction mixture. In contrast, the semicontinuous strategy maintained lower and prolonged concentrations of H(2)O(2), which allowed a clearly greater decolorization (48% after 2 h). In addition, the effect of Mn(+2) concentration on the decolorization efficiency was investigated to establish the optimal application of the MnP-oxidative system. The enzymatic treatment provoked not only the destruction of the chromophoric groups but also a noticeable breakdown of the chemical structure of the dye. In experiments with pure enzyme, MnP proved to be the main factor responsible for the dye decolorization.