仿酶催化木素降解研究进展

综述了近年来仿酶催化木素降解的研究进展,重点介绍了木素降解的机理和产物形成做了概述总结,并对仿酶催化剂进行了介绍归纳,包括Fenton催化剂、Salen配合物、金属卟啉配合物和GIF体系等。简要分析了木素降解的机理,并给出采用不同仿酶催化剂降解木素的代表性案例。鉴于传统的木素降解方法条件较为苛刻且不符合环保要求,为了使木素在温和环保的条件中降解,文中专门对仿酶催化剂的处理条件作了较详细的介绍。最后展望了仿酶催化木素降解的发展前景。对木素降解研究工作具有一定参考价值。     

仿生酶菌协同体系预处理木质素机理及特性

白蚁-真菌自然共生体系可有效转化木质纤维素类生物质,其本质在于对木质素物理结构的破坏和官能团的修饰,减少木质素对酶的非生产性吸附,从而提升酶解糖化效率,为生物质高效能源化利用提供新思路。本文基于白蚁肠道中存在的分解木质素酚类单元的漆酶（La）和蚁巢内降解木质纤维素的蚁巢伞菌（Te）,构建La和Te协同预处理木质素体系,比较La和典型的木质素降解菌黄孢原毛平革菌（PC）对木质素模型化合物碱木素的预处理特性。结果表明,在降解La预处理的碱木素过程中,Te产生的漆酶（La）和木质素过氧化物酶（LiP）活性最大值较未处理的碱木素样品分别提升43.3%、58.5%,PC产生的漆酶（La）和锰过氧化物酶（MnP）活性最大值较未处理的碱木素样品分别提升35.9%、31.6%。漆酶预处理强化了Te、PC对碱木素官能团的修饰和物理结构的破坏。傅里叶红外转换光谱分析（FTIR）表明酶菌协同体系处理后碱木素特征官能团的吸收峰显著降低。扫描电镜（SEM）和压汞测试结果表明酶菌协同体系对碱木素表面结构破坏严重,La和Te协同（La+Te）体系预处理后的碱木素平均孔径比单一La和单一Te分别显著提升31.1%、45.6%。经La+Te体系预处理后的碱木素最大酶吸附量较未处理的碱木素减少了51.5%,由于非生产性吸附显著减少,后续纤维素酶的转化率较未处理的碱木素样品提高了71.5%。本文证明了通过漆酶与真菌协同作用可有效改变碱木素物化特性,从而有效促进后续纤维素的酶解糖化,为木质纤维素类生物质高效利用提供指导。     

针叶木硫酸盐浆中残留木素的GIF体系仿酶降解机理的研究

由于针叶木硫酸盐浆中的残留木素含有大量难降解的LCC结构及其它缩合型木素结构,所以硫酸盐浆的分离木素可以用作难降解型木素大分子的一种结构模型。文章采用Cu2+/吡啶/过氧化氢组成的GIF仿酶体系对马尾松硫酸盐未漂浆的纤维素酶酶解木素(CEL)进行了仿酶降解,并对降解产物进行了分离和化学组成分析。通过FT-IR以及GC-MS测定,解析了CEL在降解过程中的结构变化,并对CEL的仿酶降解机理进行了探讨。研究结果表明:这种仿酶降解方法对浆中残留木素有较强的降解能力,一部分木素大分子被氧化降解为香草醛和香草酸等低分子化合物,使得降解后的产物中羰基和羟基增多。研究发现木素在仿酶体系中存在苯环及其侧链上C-H键的破坏、木素结构中Ca-Cb连接键的断裂、苯环的开环等反应。因此,该项研究可以为开发纸浆的仿酶漂白技术提供理论依据。     

碳源对平菇选择性降解稻草木素及其后续酶解性能的影响

前期研究发现,平菇预处理稻草表现出比较好的脱木素选择性和纤维素酶水解效果。为了使预处理后的稻草在纤维素酶水解过程中得到更多的单糖,通过向平菇预处理稻草固体培养基中添加麸皮、玉米皮和木聚糖,研究外加碳源对平菇降解稻草木素的选择性及后续酶水解效果的影响。结果表明,添加适量的麸皮和木聚糖可提高平菇处理稻草的脱木素选择性。麸皮和木聚糖添加量为稻草粉的10%时,脱木素选择系数分别由对照的1.86增加至2.58和2.03;预处理样品酶解后原料中总糖的46.8%和45.9%转化为可发酵单糖,分别比对照的35.5%提高了30%和28%。添加玉米皮对平菇预处理酶解效果影响相对较小,添加量为5%时,预处理样品酶解后总糖转化率仅比对照提高15%,增加添加量酶解总糖转化率反而低于对照样品。     

氮源浓度和芳香化合物对白腐菌Panus conchatus产木素降解酶…

白腐菌Ｐａｎｕｓｃｏｎｃｈａｔｕｓ只分泌锰过氧化的酶和漆酶，不分泌木素过氧化物酶。该菌分泌这两种酶的能力大大高分子同样培养基条件下的Ｐｌｅｕｒｏｔｕｓｓａｊｏｒ－ｃａｊｕ。氮源浓度对Ｐａｎｕｓｃｏｎｃｈａｔｕｓ分泌木素降解酶的能力有显著影响，高氮条件可能是这种类型的白腐菌生产木素降解酶的必要条件，研究不同芳香化合物以Ｐａｎｕｓｃｏｎｃｈａｔｕｓ木素降解酶分泌情况的影响发现，紫丁胺醛和香豆酸对其锰过     

白腐菌预处理对稻草化学组分及酶水解的影响

采用5株白腐菌预处理稻草,对预处理过程中产生的木质纤维素降解酶系以及稻草化学组分变化进行了分析,研究了预处理对后续纤维素酶水解效率的影响。研究结果表明,5株白腐菌在预处理期间(0~50天)均能检测到漆酶(Lac)、锰过氧化物酶(MnP)和纤维素酶(Cel)活性,但未检测到木质素过氧化物酶(LiP)活性。其中凤尾菇培养第20天Lac活性达到最高,为2244 U/L;平菇培养40天MnP活性最高,达771 U/L;凤尾菇和平菇的木质素降解选择性指数(SI)随着预处理时间延长呈上升趋势,培养至50天时平菇的SI达到1.87,比其它4株白腐菌表现出更好的选择性降解木质素能力。云芝4号、平菇和凤尾菇表现出良好的预处理效果,经此3菌株预处理50天的稻草粉,在每克底物20 FPU酶用量条件下用纤维素酶水解48 h,酶水解总糖转化率分别达到59.6%、56.3%和54.4%。     

氮源浓度和芳香化合物对白腐菌Panus conchatus产木素降解酶的影响

白腐菌Panusconchatus只分泌锰过氧化物酶和漆酶，不分泌术素过氧化物酶，该菌分泌这两种酶的能力大大高于同样培养基条件下的Pleuronssajor-caju。氮源浓度对Panusconchatus分泌术素降解酶的能力有显著影响，高氮条件可能是这种类型的白腐菌生产木素降解酶的必要条件。研究不同芳香化合物对Panusconchatus木素降解酶分泌情况的影响发现，紫丁香醇和香豆酸对其锰过氧化物酶和漆酶分别起显著的诱导作用。     

真菌细胞溶壁酶的分离纯化及其酶学性质研究

采用长梗木霉(Trichoderma Longibrachiatum Rifai)958-11菌株,经过发酵,分离提纯出一种真菌细胞溶壁酶,并对其进行酶学性质研究。长梗木霉培养,利用:(NH4)2SO4沉淀,sartobind S离子交换层析和Sephacryl S-200柱层析对该发酵液进行分离纯化,用担子菌做为酶反应的底物研究该酶的酶学性质。经发酵获得的酶液,可在最佳反应条件下,1～2h内产出原生质体105～106个/毫升酶液。酶反应最适温度为30℃,最适pH为5.4,可以在0.6mol/L的氯化钾或0.6mol/L的硫酸镁为等渗液的条件下,溶解部分担子菌的细胞壁,得到高活力的原生质体。     

自水解预处理对稻草化学成分及酶解性能的影响

研究了自水解处理对稻草秸秆主要化学成分及酶解糖化效率的影响。结果显示:在100～160℃下对稻草进行自水解预处理,酸溶木质素的脱除程度随着自水解温度的升高而增大,而Klason木质素含量几乎没有变化,几乎全部SiO2仍然保留在预处理后草片中;稻草高聚糖的降解程度随着自水解温度的升高而增加,但由于自水解液酸性较弱,大量高聚糖仍保留在草片中;自水解预处理有利于促进稻草的酶解糖化,随着自水解预处理温度的升高和酶用量的增大,酶解液中各种聚糖得率均有不同程度的提高,但自水解温度的影响显得更为重要;经160℃自水解预处理的稻草在40 FPU/g混合酶用量下,葡聚糖和木聚糖的总转化率约为68%和45%,总糖转化率近60%。     

黄孢原毛平革菌合成的木质素过氧化物酶、锰过氧化物酶及其菌球在染料脱色过程中的作用

在黄孢原毛平革菌(Phanerochaete chrysosporium)限氮浸没式培养中，获得了含木质素过氧化物酶(LiP)和锰过氧化物酶(MnP)、只含LiP或MnP以及无LiP和MnP的4种培养物，考察了LiP，MnP及菌球在染料脱色过程中的作用. 结果表明，4种培养物对6种染料都有明显的脱色效果，甲基橙、橙I、次甲基蓝脱色率在90%左右，刚果红、直接湖蓝脱色率在80%左右，结晶紫脱色率在60%左右. 无酶情况下染料的脱色主要是菌球的吸附，有酶情况下染料的脱色主要是酶的降解. 菌球在脱色过程中除了吸附染料外，还起到提供H2O2和藜芦醇的作用，促进了染料的降解.     

一株青霉发酵产生木素过氧化物酶的工艺条件

研究了一株青霉菌P6 (Penicillium sp. P6)液体发酵产生木素过氧化物酶的工艺条件,考察了青霉菌P6在天然培养基稻米糠、玉米糠、麦糠和合成培养基GMY中的产酶情况,结果表明,麦糠是较理想的底物. 通过单因素方法研究了添加苯甲醇和MnSO4对酶活的影响;采用正交实验对MnSO4添加量、麦糠含量和培养基pH进行了研究,在实验范围内最佳酶活达到了2.69 U/mL. 进一步通过单双因素回归实验研究了麦糠和MnSO4的最佳含量,得到了最佳培养条件为孢子接种量1.1×106 mL-1,培养时间8 d,500 mL三角瓶装液量150 mL. 最佳培养基组成为麦糠50 g/L,初始pH 4.8, MnSO4 5.66 mmol/L.     

Comparing Ligninolytic Capabilities of Bacterial and Fungal Dye-Decolorizing Peroxidases and Class-II Peroxidase-Catalases

We aim to clarify the ligninolytic capabilities of dye-decolorizing peroxidases (DyPs) from bacteria and fungi, compared to fungal lignin peroxidase (LiP) and versatile peroxidase (VP). With this purpose, DyPs from Amycolatopsis sp., Thermomonospora curvata, and Auricularia auricula-judae, VP from Pleurotus eryngii, and LiP from Phanerochaete chrysosporium were produced, and their kinetic constants and reduction potentials determined. Sharp differences were found in the oxidation of nonphenolic simple (veratryl alcohol, VA) and dimeric (veratrylglycerol-β- guaiacyl ether, VGE) lignin model compounds, with LiP showing the highest catalytic efficiencies (around 15 and 200 s⁻¹·mM⁻¹ for VGE and VA, respectively), while the efficiency of the A. auricula-judae DyP was 1–3 orders of magnitude lower, and no activity was detected with the bacterial DyPs. VP and LiP also showed the highest reduction potential (1.28–1.33 V) in the rate-limiting step of the catalytic cycle (i.e., compound-II reduction to resting enzyme), estimated by stopped-flow measurements at the equilibrium, while the T. curvata DyP showed the lowest value (1.23 V). We conclude that, when using realistic enzyme doses, only fungal LiP and VP, and in much lower extent fungal DyP, oxidize nonphenolic aromatics and, therefore, have the capability to act on the main moiety of the native lignin macromolecule.     

Freeze‐drying of lignin peroxidase: influence of lyoprotectants on enzyme activity and stability

The effect of different lyoprotectants (sucrose, dimethyl‐succinate buffer (DMS), bovine serum albumin (BSA), mannitol and dextran, mw 60 kDa) on the stability of the enzyme lignin peroxidase (LiP, EC number: 1.11.1.‐), both during the freeze‐drying process and storage were investigated. The shelf stability tests were performed at 4 °C and 27 °C. Both DMS buffer and sucrose showed a good protective action: the former was particularly effective during the process, while the latter improved the stability during storage. In contrast, mannitol and dextran had negative effects, reducing the activity also in the lignin peroxidase solution. BSA was discarded because, in the range of compatible concentrations with LiP, it does not confer a consistent structure to the freeze‐dried product. © 2002 Society of Chemical Industry     

曝气式反应器中木质素过氧化物酶的合成及对印染废水配制液的脱色处理

比较了黄孢原毛平革菌在3种生物反应器(搅拌式反应器、鼓泡式反应器、曝气式反应器)中合成木质素过氧化物酶(LiP)和锰过氧化物酶(MnP)的差异. 结果表明，曝气式反应器对酶的合成(尤其是LiP)最为有利. 考察了曝气式反应器中半连续培养和连续培养两种方式下酶的合成和橙I脱色情况，发现半连续培养可使培养体系长时间保持较高酶活力，置换比例为1/2时染料废水可连续脱色5批，脱色率达到90%以上，比脱色率在46.7 g/(g×d)以上. 连续培养条件下酶很快失活，废水的脱色率迅速下降. 在曝气式反应器中用半连续培养的方式(置换比例1/2)对实际印染废水进行处理，可处理废水4批，前3批脱色率达到90%以上，第4批有明显下降.     

Decolorization of Direct Blue GLL with enhanced lignin peroxidase enzyme production in Comamonas sp UVS

BACKGROUND: The present work aims to study the production of lignin peroxidase (LiP) enzyme by Comamonas sp UVS using various media, and lignocellulosic waste materials, and its effect on decolorization of Direct Blue GLL (DBGLL). RESULTS: Yeast extract medium was found to be more effective for the production of LiP and also for the decolorization of DBGLL. The bagasse powder along with yeast extract induced LiP activity. Comamonas sp UVS decolorized DBGLL dye (50 mg L^?1) within 13 h at static condition in YE broth. It could degrade up to 300 mg L^?1 of dye within 55 h. The maximum rate (Vmax) of decolorization was 12.41 ± 0.55 mg dye g cell^?1 h^?1 with the Michaelis constant (Km) value as 6.20 ± 0.27 mg L^?1. The biodegradation was monitored by UV‐Vis, GC‐MS and HPLC. CONCLUSION: The use of agricultural by‐products for the activity enhancement of the ligninolytic enzymes is a cost effective process. It also resolves the problem of the disposal of agro‐residues. This system can be applied for the degradation of different recalcitrant compounds. Copyright © 2008 Society of Chemical Industry     

关于木素生物降解酶类活力测定问题的讨论

由于木素结构和生物降解过程复杂多样，全面测定一个菌株的木素降解活力也比较困难，目前已发现三类主要参与木素降解的酶类（LIP、MnP、漆酶），但至今很少有国际上公认的酶活力测定方法，也未见到能把某一菌株木素降解酶类的活力与其在木质纤维材料中降解木素的能力定量关联的报导。本文综合评述了几类主要的木素降解酶类活力测定方法的作用机制和存在的问题，指出一个菌株的木素降解活力是由它的生理特性和总体代谢能力共同决定的，仅由一类或几类酶活力的表现水平难以对其作出确切的评价，从这个角度出发进行研究才能更客现他认识菌株的木素降解活力．     

Black liquor detoxification by laccase of Trametes versicolor pellets

Black liquors from a soda pulping mill were treated with the white‐rot fungus Trametes versicolor to detoxify and reduce colour, aromatic compounds and chemical oxygen demand (COD). The fungus was used in the form of pellets in aerated reactors (fluidized, stirred and air‐pulsed reactors). Reductions in colour and aromatic compounds of 70–80% and in COD of 60% were achieved. During the different experiments, laccase activity was detected but neither lignin peroxidase (LiP) nor manganese peroxidase activities were detected, although T versicolor is able to produce these enzymes. Experiments also showed a LiP activity inhibitory effect produced by lignin. From the results obtained, it can be concluded that there is a relationship between laccase production and toxicity reduction. This correlation responds to the equation Laccase production = 1.57 LN (toxicity reduction) ?16.40. Copyright © 2003 Society of Chemical Industry     

固定化黄孢原毛平革菌木素过氧化物酶的研究

用大孔吸附树脂进行黄孢原毛平革菌来源的木素过氧化物酶固定化试验,筛选出固定化效果较好的XAD7HP大孔树脂,研究了其固定化条件。结果表明,当树脂1.0g,酶液pH4.5,加酶量87.2U,吸附温度25℃,吸附4h,戊二醛质量分数0.2%,戊二醛处理时间120min,可获得最佳的固定化效果,固定化酶活力可达到16U/g(对载体)。     

木质素基二氧化硅复合纳米颗粒的制备及在高密度聚乙烯中的应用

针对目前木质素基SiO₂复合纳米颗粒聚集严重及木质素负载量低,难以应用的现状,以碱木质素为主要原料,先通过磷酸化改性制备磷酸化碱木质素,再利用酸析共沉法将1.2份磷酸化碱木质素与1份纳米SiO₂(均为质量份)复合制备了木质素-SiO₂复合纳米颗粒,并探究复合颗粒对高密度聚乙烯(HDPE)力学性能的影响。FT-IR、XPS、TEM、TG和静态接触角测试结果表明,木质素主要以氢键作用与SiO₂结合;与原料二氧化硅相比,复合颗粒的粒径从25 nm增加到40 nm,聚集程度明显减弱;复合纳米颗粒中木质素占47%(质量分数);表面的疏水性增强,有利于复合颗粒在高密度聚乙烯中均匀分散,显著提高了HDPE的拉伸强度。与碱木质素/HDPE复合材料相比,木质素-SiO₂复合纳米颗粒/HDPE复合材料的拉伸强度和断裂拉伸率分别提高了48.68%和73.57%。     

Improvement of the catalytic performance of lignin peroxidase in reversed micelles

BACKGROUND: Anionic surfactant sodium bis (2‐ethylhexyl) sulfosuccinate (AOT) had an inhibiting effect on lignin peroxidase (LiP). To improve the catalytic activity of LiP in an AOT reversed micelle in isooctane, nonionic surfactant polyoxyethylene lauryl ether (Brij30) was incorporated into the interfacial membrane. H₂O₂ played dual roles in the LiP‐catalyzed oxidation of substrates. To obtain a sustainable high activity of LiP, a coupled enzymatic reaction, i.e. the glucose oxidase (GOD)‐catalyzed oxidation of glucose was used as an H₂O₂ source. RESULTS: Owing to modification of the charge density of the interfacial membrane, the activity of LiP in an optimized AOT/Brij30 reversed micellar medium (χ_B (the molar percentage of Brij30) = 0.53, ω₀ ([H₂O]/([AOT] + [Brij30]) = 23, pH = 4.8) was 40 times that in a single AOT reversed micelle. Due to the controlled release of H₂O₂, the concentration of H₂O₂ in the mixed reversed micellar medium was maintained at a moderately high level throughout, which made the LiP‐catalyzed oxidation of substrates proceed at a higher conversion rate than counterparts in which H₂O₂ was supplied externally in one batch at the beginning of the reaction. Decolourization of two waterless‐soluble aromatic dyes (pyrogallol red and bromopyrogallol red) using LiP coupled with GOD in the medium also demonstrated that a higher decolourization percentage was obtained if H₂O₂ was supplied enzymatically. CONCLUSION: The proposed measures (both physicochemical and biochemical) were very effective, giving significant improvement in the catalytic performance of LiP in a single AOT reversed micelle in isooctane, which helped to degrade or transform hydrophobic aromatic compounds with LiP in reversed micelles more efficiently. Copyright © 2007 Society of Chemical Industry