疣孢漆斑菌Myrothecium verrucaria GH-01漆酶的纯化和酶学性质研究

疣孢漆斑菌具有生长周期短,分泌漆酶酶活高等特点。利用已分离得到的疣孢漆斑菌GH-01(Myrothecium verrucaria GH-01)发酵产生粗酶液。进而通过分级沉淀、透析和层析的方法对漆酶粗酶液进行纯化。SDS-PAGE和Native-PAGE结果表明纯化可获得具有漆酶活性的单体蛋白。酶学性质研究表明,该漆酶催化最适温度为40℃,在底物ABTS存在条件下的最适p H值为4.0,且在低温及碱性条件具有较好的稳定性。此外通过漆酶对4大类染料脱色能力的研究,发现该漆酶对偶氮类的橙黄Ⅰ和蒽醌类的茜素红脱色有较好的脱色效果,反应1 h脱色率达80%以上;对三苯甲烷类的碱性品红脱色能力较弱,脱色率只能达到20%左右;脱色率最差的是杂环类的亚甲基蓝。在含10 U漆酶的体系中,对50 mg/L的染料降解效果相对最佳。     

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%。     

灵芝漆酶催化直接耐晒翠蓝GL脱色条件的优化

采用灵芝菌株发酵所得的漆酶, 对酞菁类染料直接耐晒翠蓝GL进行了催化脱色实验, 确定了脱色反应的最适条件。结果表明: 单独使用灵芝漆酶粗酶液对直接耐晒翠蓝GL具有很好的脱色效果。其最适脱色pH为3.0, 最适脱色温度为40°C, 最适漆酶用量是40 U/mL, 最适染料浓度为60 mg/L。以上述最适脱色条件对直接耐晒翠蓝GL进行脱色实验, 反应70 min, 脱色率可达94.3%。研究结果显示, 所试灵芝漆酶在印染废水治理方面具有良好的应用前景。     

漆酶高产工程菌构建及漆酶对RBBR的脱色作用

研究提取产漆酶白腐菌 (Fomelignosus)的总RNA ,利用RT PCR克隆到漆酶的cDNA ,并将其克隆到表达载体pGAPZA ,重组质粒经线性化、电激转化PichiapastorisGS115、通过底物显色反应筛选漆酶生产工程菌株 ,在最适培养条件下该菌株产酶活力高达 9 0 3U·mL-1。纯化得到漆酶对RBBR(RemazolbrilliantblueR)有很好的脱色作用 ,该酶的最适脱色pH为 5 0 ,最适脱色温度为 30℃。当溶液中漆酶活力为 1 0U·mL-1,在最适脱色条件下作用12h ,10 0mg·L-1RBBR溶液脱色率可达 90 %以上。     

一色齿毛菌漆酶的酶学特性及染料脱色研究

染料由于具有复杂的化学结构通常难以降解。本文从白腐菌一色齿毛菌LS0547中纯化出胞外漆酶并用于染料脱色实验。SDS-PAGE结果显示纯化的漆酶分子量大小为63.7kDa。漆酶氧化底物ABTS的最适pH为2.2,最适温度为50℃。叠氮钠可强烈抑制漆酶活性,半胱氨酸和二硫苏糖醇可部分抑制漆酶活性。漆酶氧化ABTS,丁香醛连氮和2,6-二甲氧基苯酚的米氏常数分别为0.217,0.306和0.199mmol/L。粗酶和纯化的漆酶用于不同化学结构的染料的脱色研究,结果表明一色齿毛菌纯化漆酶可快速对RB亮蓝进行脱色,偶氮胭脂红和结晶紫的脱色效果低于RB亮蓝,测试的三种染料均可在没有介体存在的条件下被漆酶脱色,显示出一色齿毛菌漆酶在染料废水处理中的应用前景。     

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%。     

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。     

一株产漆酶细菌的分离鉴定及酶学性质研究

[目的]分离获得产漆酶的细菌菌株,研究漆酶的酶学性质并应用于染料脱色.[方法]利用含铜的富集培养基筛选产漆酶细菌;通过形态特征、生理生化试验及16SrDNA序列分析等方法进行鉴定;以丁香醛连氮为底物测定漆酶的酶学性质;通过测定染料在最大吸收波长下吸光值的变化评价漆酶对染料的脱色效果.[结果]从森林土壤中筛选到一株漆酶高产菌株LS05,初步鉴定为解淀粉芽孢杆菌(Bacillus amyloliquefaciens);菌株LS05的芽孢漆酶以丁香醛连氮为底物的最适pH为6.6,最适温度为70℃;该酶具有较好的稳定性,经70℃处理10h或在pH 9.0条件下放置10d后可保留活性.对抑制剂SDS和EDTA具有一定的抗性,在碱性条件下可有效脱色不同的工业染料,RB亮蓝、活性黑和靛红1h内的脱色率达93％以上.[结论]Bacillus amyloliquefaciens LS05的芽孢漆酶在高温和碱性条件下稳定性强,相对于真菌漆酶具有更好的工业应用特性,可有效用于工业染料废水的处理.     

应用Coriolus vericolor菌丝球脱色染料及印染废水的研究

对白腐真菌(Coriolus vericolor)产生漆酶进行〖JP2〗了研究。发现该菌产漆酶的最适初始pH值为45。提高微量元素浓度或添加藜芦醇都可使C.versicolor的产酶能力增加,添加Tween80会有一定的抑制作用。采用C.versicolor菌丝球进行重复分批产酶试验,结果表明菌丝球的稳定性很好,同一批菌丝球可连续利用14次,平均每批酶活力可保持在672U/mL,产酶能力优于聚氨酯泡沫固定化菌丝。将粗酶液用于染料的脱色降解试验,在酶活力为3.3IU/mL〖JP〗,酸性橙浓度为500mg/L条件下,经过24h反应,脱色率达到98.5%;对含弱酸大红和卡布龙红的印染废水进行脱色试验,脱色率也达到了93%。     

白囊耙齿菌对茜素红脱色条件优化及其毒性变化检测

白囊耙齿菌Irpex lacteus是分离自倒木上的一株可以分泌漆酶和锰过氧化物酶的白腐真菌。利用I. lacteus对固体条件下的活性黑、活性红、结晶紫、茜素红和孔雀石绿进行脱色能力的检测,通过单因素和正交试验优化I. lacteus对茜素红的脱色条件,并以3种作物发芽率为指标测定茜素红被I. lacteus脱色前后的毒性变化。结果显示,I. lacteus对5种染料均可脱色,其中对茜素红染料的脱色更为彻底;单因素和正交试验优化I. lacteus对茜素红的脱色条件为：pH 7.0、葡萄糖10.0g/L、硫酸铵0.66g/L、接种量2片（Φ=8.0mm）、100.0mL三角瓶装液20.0mL,优化条件下I. lacteus对茜素红脱色10d时的脱色率为88.26%,与未优化前的脱色率相比提高了60.50%;茜素红染料被I. lacteus脱色前后毒性大小排序为：染料原液>染料脱色后>PDB培养基处理,表明茜素红染料存在一定的毒性,I. lacteus脱色茜素红后可以使其毒性减弱。通过本研究,为I. lacteus在茜素红等染料废水脱色以及降低染料废水毒性方面的应用奠定基础。     

新月弯孢霉菌丝球对染料脱色作用的研究

研究了新月弯孢霉Curvularia lunata JQH-100液体培养时产生的菌丝球对多种染料的脱色能力。结果表明,多种染料在24h内的脱色率均达到80%以上,且菌丝球稳定性良好,可重复使用6次;以菌丝球对孔雀绿脱色效果为优化指标,正交实验优化获得制备菌丝球的最佳条件为:葡萄糖20g/L、硫酸铵5g/L、马铃薯200g/L、KH2PO43g/L、MgSO45mg/L、CuSO40.5mg/L、VB15mg/L及pH5、摇床转速120r/min。在上述优化后的基础培养基(不含MgSO4、CuSO4)中分别添加微量元素Cu2+、Mn2+、Mg2+或Ca2+制备的菌丝球,对孔雀绿脱色能力增强;添加Fe2+制备的菌丝球,对孔雀绿脱色能力下降;分别添加Zn2+、Al3+或Na+制备的菌丝球对孔雀绿脱色能力与对照相近。应用优化后培养条件制备的菌丝球处理含多种染料的混合废水,也获得了较好的脱色效果。     

新月弯孢子座的结构和形成条件

新月弯孢 [Curvularialunata (Walk)Boed ]是近些年我国北方玉米主栽区新发生的弯孢菌叶斑病的致病菌 ,它以分生孢子进行重复侵染。在试验中 ,当用高粱培养基进行扩繁培养时 ,新月弯孢能产生一种无性繁殖体 子座。报道了子座产生的条件及子座的形态结构特征。     

玉米种质资源抗弯孢菌叶斑病特性研究

针对近年弯孢菌叶斑病日益严重的发生趋势,对1698份玉米种质(自交系、群体、杂交种以及特殊材料)进行了抗弯孢菌叶斑病鉴定.结果表明,中国玉米种质抗性较引进种质抗性好;不同省份所供种质抗性存在差异,北京、四川、广西种质总体抗性较好;在新选育的自交系中,鉴定出12份高抗材料;在当前培育的杂交种中,有22份高抗或抗弯孢菌叶斑病;玉米对弯孢菌叶斑病抗性在相同核基因、不同细胞质种质间无差异;玉米抗大斑病基因对抗弯孢菌叶斑病无效.     

Melanin biosynthesis in the fungus Curvularia lunata (teleomorph: Cochliobolus lunatus)

Curvularia lunata (teleomorph: Cochliobolus lunatus) is a known plant and human pathogen. Tricyclazole, a specific inhibitor of pentaketide melanin biosynthesis, blocked the biosynthesis of melanin in Curvularia lunata and caused the accumulation of the melanin metabolites flaviolin and 2-hydroxyjuglone. This showed that melanin in Curvularia lunata is produced by a pentaketide pathway from 1,8-dihydroxynaphthalene. The 1,3,8-trihydroxynaphthalene reductase (3HNR) gene, associated with the melanin pathway of Curvularia lunata, was identified and characterized. An alignment of 3HNR sequences enabled the design of primers covering conserved regions. A PCR-amplified fragment of Curvularia lunata genomic DNA was used for screening the cDNA library. Three independent cDNA clones revealed an 801-bp open reading frame encoding a 267 amino acid protein. The protein was expressed in Escherichia coli and purified to homogeneity. The predicted amino acid sequence of the 28.6-kDa protein demonstrated homology to other fungal 3HNR and other members of the short-chain dehydrogenase super family. Northern analyses revealed that 3HNR from Curvularia lunata is expressed synchronously with melanization after 3 days of Curvularia lunata growth in malt extract medium. No 3HNR reductase gene expression nor melanization was observed when Curvularia lunata was grown in yeast nitrogen base medium.     

Evaluation of a new fungus <Emphasis Type="Italic">Ceriporia lacerate</Emphasis> strain P2—its ability to decolorize Alizarin Red and Methyl Orange

A new fungus Ceriporia lacerate P2 which belongs to family Polyporaceae was evaluated for its ability to decolorize two different dyes Alizarin Red and Methyl Orange. Different parameters such as incubation time, pH, carbon source, nitrogen source and carbon/nitrogen regimes were used to find out the optimum medium for Ceriporia lacerate P2 on its ability of decolorization. The results show that the fungus had different ability to decolorize the two tested dyes. For Alizarin Red, the most suitable medium was at pH 3 and the best carbon and nitrogen source were sucrose and ammonium nitrate. While for Methyl Orange, the optimum medium was at pH 7–9 and the best carbon and nitrogen source were sucrose and urea.     

Decolorization of simulated textile dye baths by crude laccases from Trametes hirsuta and Cerrena unicolor

In this study crude laccases from the white‐rot fungi Cerrena unicolor and Trametes hirsuta were tested for their ability to decolorize simulated textile dye baths. The dyes used were Remazol Brilliant Blue R (RBBR) (100 mg/L), Congo Red (12.5 mg/L), Lanaset Grey (75 mg/L) and Poly R‐478 (50 mg/L). The effect of redox mediators on dye decolorization by laccases was also assessed. C. unicolor laccase was able to decolorize all the dyes tested. It was especially effective towards Congo Red and RBBR with 91 and 80% of color removal in 19.5 h despite the fact that simulated textile dye baths were used. Also Poly R‐478 and Lanaset Grey were partially decolorized (69 and 48%, respectively). C. unicolor laccase did not need any mediators for removing the dyes. However, T. hirsuta laccase was only able to decolorize simulated Congo Red and RBBR dye baths (91 and 45%, respectively) in 19.5 h without mediators. When using mediators the decolorization capability was enhanced substantially, e.g. Poly R‐478 was decolorized by 78% in 25.5 h. On the whole, both laccases showed potential to be used in industrial applications.     

Enlargement of anode for enhanced simultaneous azo dye decolorization and power output in air-cathode microbial fuel cell

Air-cathode, microbial fuel cells (MFC) with different anode surface areas were evaluated for simultaneous decolorization of Congo Red and bioelectricity production. Doubling the anode area from 18 to 36?cm² increased net power by 150?% (0.16–0.4?mW), normalized power (per anode surface area) by 22?% (88–107?mW?m^?2) and Congo Red decolorization by 163?% (1.6–4.2?mg?l^?1?h^?1). Quadrupling the original anode area induced an additional 5?% increase (up to 4.2?mW) in net power and 174?% increase (up to 11.5?mg?l^?1?h^?1) in Congo Red decolorization; however, normalized power decreased by 85?% (down to 58?mW?m^?2). Increased bacterial attachment could account for both the enhanced power and Congo Red decolorization in larger anode MFCs. The limited effect on power output likely arises from cathode limitation or inefficient utilization of anodes.     

Decolorization of Alizarin Red and other synthetic dyes by a recombinant laccase from Pichia pastoris

A cDNA encoding for a laccase was isolated from the white-rot fungus Lenzites gibbosa by RT-PCR and expressed in the Pichia pastoris. The laccase native signal peptide efficiently directed the secretion of the recombinant laccase in an active form. Factors influencing laccase expression, such as pH, cultivation temperature, copper concentration and methanol concentration, were optimized. The recombinant enzyme was purified to electrophoretic homogeneity, and was estimated to have a MW of ~61.5 kDa. The purified enzyme behaved similarly to the native laccase produced by L. gibbosa and efficiently decolorized Alizarin Red, Neutral Red, Congo Red and Crystal Violet, without the addition of redox mediators. The decolorization capacity of this recombinant enzyme suggests that it could be a useful biocatalyst for the treatment of dye-containing effluents. This study is the first report on the synthetic dye decolorization by a recombinant L. gibbosa laccase.     

Biodegradation of azo and heterocyclic dyes by Phanerochaete chrysosporium.

Biodegradation of Orange II, Tropaeolin O, Congo Red, and Azure B in cultures of the white rot fungus, Phanerochaete chrysosporium, was demonstrated by decolarization of the culture medium, the extent of which was determined by monitoring the decrease in absorbance at or near the wavelength maximum for each dye. Metabolite formation was also monitored. Decolorization of these dyes was most extensive in ligninolytic cultures, but substantial decolorization also occurred in nonligninolytic cultures. Incubation with crude lignin peroxidase resulted in decolorization of Azure B, Orange II, and Tropaeolin O but not Congo Red, indicating that lignin peroxidase is not required in the initial step of Congo Red degradation.     

Biodegradation of azo and heterocyclic dyes by Phanerochaete chrysosporium

Biodegradation of Orange II, Tropaeolin O, Congo Red, and Azure B in cultures of the white rot fungus, Phanerochaete chrysosporium, was demonstrated by decolarization of the culture medium, the extent of which was determined by monitoring the decrease in absorbance at or near the wavelength maximum for each dye. Metabolite formation was also monitored. Decolorization of these dyes was most extensive in ligninolytic cultures, but substantial decolorization also occurred in nonligninolytic cultures. Incubation with crude lignin peroxidase resulted in decolorization of Azure B, Orange II, and Tropaeolin O but not Congo Red, indicating that lignin peroxidase is not required in the initial step of Congo Red degradation.