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%。     

出芽短梗霉胞外酸性漆酶

通过愈创木酚法平板检测10株出芽短梗霉,发现5株菌能够分泌胞外多酚氧化酶,反应最适pH在2.0左右,均属于酸性多酚氧化酶。菌株NG的酶活最高,达110 U/mL。添加H2O2、EDTA以及过氧化氢酶不显著影响菌株NG胞外酶活,表明NG分泌的多酚氧化酶中不含有锰过氧化物酶（MnP）和不依赖Mn2＋的过氧化物酶（MiP）,属于漆酶（Lac）。     

灵芝漆酶催化阳离子红2GL脱色的研究

真菌漆酶在纺织物染料脱色及其废水净化等领域有着巨大的应用潜力。阳离子红2GL是使用广泛又难以处理的一种染料,现有的方法治理效果差。本研究优化了灵芝漆酶催化阳离子红2GL脱色的主要工艺参数:最适pH、温度、ABTS用量、漆酶用量和染料浓度分别为4.5、20℃、0.083mmol/L、10U/mL和50mg/L。在所得的最优条件下反应30min,阳离子红2GL的脱色率可达90.3%;反应24h,脱色率达100.0%。     

变色栓菌产锰过氧化物酶的条件优化

研究了多种培养基组分及培养条件对变色栓菌产锰过氧化物酶(MnP)的影响.当培养基中果糖浓度为20g/L,酒石酸铵浓度为10mmoL/L,吐温80浓度为1.0g/L,MgSO4·7H2O为0.43g/L,最终pH为4.5,500mL三角瓶装液量为100mL,接种量为10片(φ8mm)菌苔,培养温度为30℃,转速为280r/min时,MnP的活力有了很大程度的提高,最高酶活力可达2,270U/L.     

白腐真菌处理灰法造纸黑液废水的研究

研究了不同白腐真菌菌株对灰法造黑液废水的处理,考察了黑液废水浓度和碳氮源添加量对黑液脱色及COD去除率的影响。研究表明,变色栓菌（Trametes verscolor)对黑液废水的处理效果最好,其COD去除率为64．25％,脱色率为47．31％,用自选的白腐真菌AH28－2菌株处理未经稀释的黑液废水,分别添加0．2％纤维二维糖和0．02％天冬酰胺,COD去除率达62．45％和68．60％,研究发现锰过氧化物酶（MnP)和木素过氧化物酶（LiP)对COD去除率有直接影响,MnP/LiP酶活力值越高,处理效果越好。     

锰过氧化物酶(MnP)的研究进展

综述了国内外锰过氧化物酶（MnP)研究的最新动态。MnP是一种含有血红素的过氧化物酶,仅在白腐菌中发现。从产生MnP的微生物、MnP对Mn^2 的依赖性、催化机制、蛋白质研究进展、基因结构和基因表达的研究以及工业应用前景方面进行了系统概述。     

利用农业废弃物固态发酵裂褶菌F17产锰过氧化物酶的基质优化(英文)

锰过氧化物酶(MnP)在环保领域有着广阔的应用前景。目前,利用廉价基质生产MnP,尤其是利用工农业废弃物生产MnP的研究受到了国内外学者的广泛关注。本实验利用响应面方法从几种不同的农业废弃物中筛选裂褶菌F17(Schizophyllum sp.F17)产MnP的固态发酵基质。结果表明,以0.52∶0.15∶0.33的比例组成的松木屑、稻草和黄豆粉的混合基质为发酵产MnP的最佳基质,发酵第6天MnP的活力最高,达到11.18 U/g。因此,利用农业废弃物固态发酵产锰过氧化物酶在减低酶的成本和环境污染物治理方面具有重要的意义。     

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.     

藜芦醇和吐温80对白腐菌产木质素降解酶的影响及在偶氮染料脱色中的作用

担子菌PM2在限氮液体培养下,分泌木质素过氧化物酶和锰过氧化物酶;藜芦醇、吐温 80的补充,提高了该菌锰过氧化物酶的产生,获得的最大锰过氧化物酶Mnp酶活为254.2u/L、190.2 u/L,分别是对照的3.4倍和2.5倍。选择三种偶氮染料,在染料体系下,进一步分析藜芦醇、吐温 80对担子菌PM2产过氧化物酶及染料脱色的影响。结果表明,担子菌PM2分泌的锰过氧化物酶Mnp与染料脱色有关,脱色程度受其分子结构特征影响;吐温80的补充,更有利于染料的脱色降解,48h后三种染料均可达到80%以上的脱色率。     

Mn-dependent peroxidase from the lignin-degrading white rot fungus Phlebia radiata

A homogeneous Mn-dependent peroxidase (MnP) was purified from the extracellular culture fluid of the lignin-degrading white rot fungus Phlebia radiata by anion exchange chromatography. The enzyme had a molecular weight of 49,000 and pI 3.8. It was a glycoprotein, containing carbohydrate moieties accounting for 10% of the molecular weight. Mn-peroxidase was capable of oxidizing phenolic compounds in the presence of H2O2, whereas the effect on nonphenolic lignin model compounds was insignificant. MnP contained protoporphyrin IX as a prosthetic group. During enzymatic reactions H2O2 converted the native MnP to compound II. Mn2+ was essential in completing the catalytic cycle by returning the enzyme to its native state. The oxidation of ultimate substrates was dependent on superoxide radicals, O2- and probably on Mn3+ generated during the catalytic cycle. MnP exhibited high activity of NADH oxidation without exogenously added H2O2. It was shown to produce H2O2 at the expense of NADH.     

Oxidative degradation of azo dyes by manganese peroxidase under optimized conditions

The application of enzyme-based systems in waste treatment is unusual, given that many drawbacks are derived from their use, including low efficiency, high costs and easy deactivation of the enzyme. The goal of this study is the development of a degradation system based on the use of the ligninolytic enzyme manganese peroxidase (MnP) for the degradation of azo dyes. The experimental work also includes the optimization of the process, with the objective of determining the influence of specific physicochemical factors, such as organic acids, H(2)O(2) addition, Mn(2+) concentration, pH, temperature, enzyme activity and dye concentration. A nearly total decolorization was possible at very low reaction times (10 min) and at high dye concentration (up to 1500 mg L(-)(1)). A specific oxidation capacity as high as 10 mg dye degraded per unit of MnP consumed was attained for a decolorization higher than 90%. Among all, the main factor affecting process efficiency was the strategy of H(2)O(2) addition. The continuous addition at a controlled flow permitted the progressive participation of H(2)O(2) in the catalytic cycle through a suitable regeneration of the oxidized form of the enzyme, which enhanced both the extent and the rate of decolorization. It was also found that, in this particular case, the presence of a chelating organic acid (e.g., malonic) was not required for an effective operation. Probably, Mn(3+) was chelated by the dye itself. The simplicity and high efficiency of the process open an interesting possibility of using of MnP for solving other environmental problems.     

Purification and characterization of manganese peroxidase of the white-rot fungus Irpex lacteus

The production of manganese peroxidase (MnP) by Irpex lacteus, purified to electrophoretic homogeneity by acetone precipitation, HiPrep Q and HiPrep Sephacryl S-200 chromatography, was shown to correlate with the decolorization of textile industry wastewater. The MnP was purified 11.0-fold, with an overall yield of 24.3%. The molecular mass of the native enzyme, as determined by gel filtration chromatography, was about 53 kDa. The enzyme was shown to have a molecular mass of 53.2 and 38.3 kDa on SDS-PAGE and MALDI-TOF mass spectrometry, respectively, and an isoelectric point of about 3.7. The enzyme was optimally active at pH 6.0 and between 30 and 40 degrees C. The enzyme efficiently catalyzed the decolorization of various artificial dyes and oxidized Mn (II) to Mn (III) in the presence of H(2)O(2). The absorption spectrum of the enzyme exhibited maxima at 407, 500, and 640 nm. The amino acid sequence of the three tryptic peptides was analyzed by ESI Q-TOF MS/MS spectrometry, and showed low similarity to those of the extracellular peroxidases of other white-rot basidiomycetes.     

Mn(II) oxidation is the principal function of the extracellular Mn-peroxidase from Phanerochaete chrysosporium

The manganese peroxidase (MnP), from the lignin-degrading fungus Phanerochaete chrysosporium, an H2O2-dependent heme enzyme, oxidizes a variety of organic compounds but only in the presence of Mn(II). The homogeneous enzyme rapidly oxidizes Mn(II) to Mn(III) with a pH optimum of 5.0; the latter was detected by the characteristic spectrum of its lactate complex. In the presence of H2O2 the enzyme oxidizes Mn(II) significantly faster than it oxidizes all other substrates. Addition of 1 M equivalent of H2O2 to the native enzyme in 20 mM Na-succinate, pH 4.5, yields MnP compound II, characterized by a Soret maximum at 416 nm. Subsequent addition of 1 M equivalent of Mn(II) to the compound II form of the enzyme results in its rapid reduction to the native Fe3+ species. Mn(III)-lactate oxidizes all of the compounds which are oxidized by the enzymatic system. The relative rates of oxidation of various substrates by the enzymatic and chemical systems are similar. In addition, when separated from the polymeric dye Poly B by a semipermeable membrane, the enzyme in the presence of Mn(II)-lactate and H2O2 oxidizes the substrate. All of these results indicate that the enzyme oxidizes Mn(II) to Mn(III) and that the Mn(III) complexed to lactate or other alpha-hydroxy acids acts as an obligatory oxidation intermediate in the oxidation of various dyes and lignin model compounds. In the absence of exogenous H2O2, the Mn-peroxidase oxidized NADH to NAD+, generating H2O2 in the process. The H2O2 generated by the oxidation of NADH could be utilized by the enzyme to oxidize a variety of other substrates.     

毛栓菌产漆酶条件优化及该酶对合成染料脱色的特性

【目的】提高菌株Trametes hirsuta SYBC-L19漆酶产量,并研究该酶对合成染料脱色的性质。【方法】通过单因素和响应面设计,对产漆酶培养基进行优化。【结果】最优培养基为:玉米粉20.0 g/L、马铃薯淀粉32.4 g/L、酒石酸铵2.9 g/L、吐温80 0.5 g/L、CuSO4.5H2O 2.0 mmol/L、香兰素0.54 mmol/L、NaH2PO4.2H2O 2.0 g/L、MgSO4.7H2O0.5 g/L、MnSO4.H2O 0.1 g/L;最佳培养条件为:培养温度30°C,初始pH 6.0,装液量40 mL/250 mL,接种量8%。【结论】培养8 d酶活达35 U/mL,是优化前的39倍。对漆酶催化合成染料脱色进行了考察,发现该酶在60°C下对偶氮类染料AR1和RB5能迅速脱色,5 min内即可完成。     

Purification and characterization of Mn-peroxidase from Musa paradisiaca (banana) stem juice

Mn-peroxidase (MnP), a biotechnologically important enzyme was purified for the first time from a plant source Musa paradisiaca (banana) stem, which is an agro-waste easily available after harvest of banana fruits. MnP was earlier purified only from the fungal sources. The enzyme was purified from stem juice by ultrafiltration and anion-exchange column chromatography on diethylamino ethylcellulose with 8-fold purification and purification yield of 65%. The enzyme gave a single protein band in SDS-PAGE corresponding to molecular mass 43 kDa. The Native-PAGE of the enzyme also gave a single protein band, confirming the purity of the enzyme. The UV/VIS spectrum of the purified enzyme differed from the other heme peroxidases, as the Soret band was shifted towards lower wavelength and the enzyme had an intense absorption band around 250 nm. The K(m) values using MnSO4 and H2O2 as the substrates of the purified enzyme were 21.0 and 9.5 microM, respectively. The calculated k(cat) value of the purified enzyme using Mn(II) as the substrate in 50 mM lactate buffer (pH 4.5) at 25 degrees C was 6.7s(-1), giving a k(cat)/K(m) value of 0.32 microM(-1)s(-1). The k(cat) value for the MnP-catalyzed reaction was found to be dependent of the Mn(III) chelator molecules malonate, lactate and oxalate, indicating that the enzyme oxidized chelated Mn(II) to Mn(III). The pH and temperature optima of the enzyme were 4.5 and 25 degrees C, respectively. The enzyme in combination with H2O2 liberated bromine and iodine in presence of KBr and KI respectively. All these enzymatic characteristics were similar to those of fungal MnP. The enzyme has the potential as a green brominating and iodinating agent in combination with KBr/KI and H2O2.     

Immobilization of manganese peroxidase from Lentinula edodes and its biocatalytic generation of MnIII-chelate as a chemical oxidant of chlorophenols

Manganese peroxidase (MnP) purified from commercial cultures of Lentinula edodes was covalently immobilized through its carboxyl groups using an azlactone-functional copolymer derivatized with ethylenediamine and 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (EEDQ) as a coupling reagent. The tethered enzyme was employed in a two-stage immobilized MnP bioreactor for catalytic generation of chelated MnIII and subsequent oxidation of chlorophenols. Manganese peroxidase immobilized in the enzyme reactor (reactor 1) produced MnIII-chelate, which was pumped into another chemical reaction vessel (reactor 2) containing the organopollutant. Reactor 1-generated MnIII-chelates oxidized 2,4-dichlorophenol and 2,4, 6-trichlorophenol in reactor 2, demonstrating a two-stage enzyme and chemical system. H2O2 and oxalate chelator concentrations were varied to optimize the immobilized MnP's oxidation of MnII to MnIII. Oxidation of 1.0 mM MnII to MnIII was initially measured at 78% efficiency under optimized conditions. After 24 h of continuous operation under optimized reaction conditions, the reactor still oxidized 1.0 mM MnII to MnIII with approximately 69% efficiency, corresponding to 88% of the initial MnP activity.     

Oxidation of hydroquinones by the versatile ligninolytic peroxidase from Pleurotus eryngii. H2O2 generation and the influence of Mn2+.

Formation of H2O2 during the oxidation of three lignin-derived hydroquinones by the ligninolytic versatile peroxidase (VP), produced by the white-rot fungus Pleurotus eryngii, was investigated. VP can oxidize a wide variety of phenols, including hydroquinones, either directly in a manner similar to horseradish peroxidase (HRP), or indirectly through Mn3+ formed from Mn2+ oxidation, in a manner similar to manganese peroxidase (MnP). From several possible buffers (all pH 5), tartrate buffer was selected to study the oxidation of hydroquinones as it did not support the Mn2+-mediated activity of VP in the absence of exogenous H2O2 (unlike glyoxylate and oxalate buffers). In the absence of Mn2+, efficient hydroquinone oxidation by VP was dependent on exogenous H2O2. Under these conditions, semiquinone radicals produced by VP autoxidized to a certain extent producing superoxide anion radical (O2*-) that spontaneously dismutated to H2O2 and O2. The use of this peroxide by VP produced quinone in an amount greater than equimolar to the initial H2O2 (a quinone/H2O2 molar ratio of 1 was only observed under anaerobic conditions). In the presence of Mn2+, exogenous H2O2 was not required for complete oxidation of hydroquinone by VP. Reaction blanks lacking VP revealed H2O2 production due to a slow conversion of hydroquinone into semiquinone radicals (probably via autooxidation catalysed by trace amounts of free metal ions), followed by O2*- production through semiquinone autooxidation and O2*- reduction by Mn2+. This peroxide was used by VP to oxidize hydroquinone that was mainly carried out through Mn2+ oxidation. By comparing the activity of VP to that of MnP and HRP, it was found that the ability of VP and MnP to oxidize Mn2+ greatly increased hydroquinone oxidation efficiency.     

树状多节孢酸性磷酸酶的分离纯化与性质研究

树状多节孢Nodulisporium sylviforme是从东北红豆杉Taxus cuspidata分离、可产生紫杉醇的内生真菌。研究以树状多节孢为材料,利用液体发酵手段获得菌丝体,通过CM-cellulose阴离子交换柱层析、Q-Sepharose阳离子交换柱层析和FPLC凝胶过滤层析（Superdex 75）,获得纯化的树状多节孢酸性磷酸酶蛋白（Nod-ACP）。结合FPLC和SDS-PAGE分析,判定该磷酸酶为分子量44kDa单亚基蛋白。酶学性质研究表明,其最适pH值为3.0,最适温度为58℃。6