Organic Solvents Enhanced Spectrofluorimetric Method for Determination of Laccase Activity

A novel fluorimetric method for determination of laccase activity in organic solvents is proposed, based on the oxidation ofo-phenylenediamine (1,2-diaminobenzene, OPDA) catalyzed by laccase yielding 2,3-diaminophenazine. The optimal conditions for laccase in organic media areT=55°C, pH=6.5, 1.0×10?2mol/L OPDA, 1.25 mL ethanol, 1.25 mL 1,4-dioxane and 1.25 mL acetone. The linear range of the method proposed in ethanol, 1,4-dioxane and acetone media were 0.44–19.33, 0.11–20.85, 0.38–21.05 U with the detection limit of 0.088, 0.022, 0.076 U, respectively. The proposed method has been applied to the analysis of laccase activity of real samples with more accurate and sensitive than that of the previous method reported.

Optimization of Culture Components for Laccase Activity from Pleurotus Ostreatus P40 Using Response Surface Methodology

High enzymatic activity is required for laccase applications.Central composite design （CCD）-based response surface methodology （RSM） can effectively increase the enzymatic activity of Pleurotus ostreatus P40 in liquid substrate fermentation.Initial screening of the nutritional components was performed using a Plackett-Burman design.The variables,namely,bran,bagasse,Tween 80,and yeast extract,were found to have statistically significant effects on laccase activity.These variables were further optimized using CCD-based RSM.Optimal concentrations for the maximum laccase activity were 8.144 2 g/L bran,50 g/L bagasse,0.424 1 mL/L Tween 80,and 2.832 5 g/L yeast extract.Under optimized conditions,the maximum measured laccase activity reached 96 480 U/L,which was close to the predicted value （104 830 U/L） by RSM.Therefore,RSM can be used to optimize culture components for laccase activity from Pieurotus ostreatus P40.

Preparation of Magnetic Chitosan Nanoparticles and Immobilization of Laccase

The magnetic chitosan nanoparticles were prepared by reversed-phase suspension method using Span-80 as an emulsifier, glutaraldehyde as cross-linking reagent. And the nanoparticles were characterized by TEM, FT-IR and hysteresis loop. The results show that the nanoparticles are spherical and almost superparamagnetic. The laccase was immobilized on nanoparticles by adsorption and subsequently by cross-linking with glutaraldehyde. The immobilization conditions and charac-terizations of the immobilized laccase were investigated. The optimal immobilization conditions were as follows： 10 mL of phosphate buffer （0.1 M, pH 7.0） containing 50 mg of magnetic chitosan nanoparticles, 1.0 mg·mL-1 of laccase and 1% （v/v） glutaraldehyde, immobilization temperature of 4 ℃ and immobilization time of 4 h. The immobilized laccase exhibited an appreciable catalytic capability （480 units·g-1 support） and had good storage stability and operation stability. The Km of immobilized and free laccase for ABTS were 140.6 and 31.1 μM in phosphate buffer （0.1 M, pH 3.0） at 37 ℃, respectively. The immobilized laccase is a good candidate for the research and development of biosensors based on laccase catalysis.

Immobilization of laccase on amine-terminated magnetic nano-composite by glutaraldehyde crosslinking method

The immobilizing procedure has great influence on the activity of the immobilized laccase. The laccase was immobilized on copper tetraaminophthalocyanine (CuTAPc)-Fe3O4 magnetic nano-composite by glutaraldehyde crosslinking method via a two-step reaction. For 1 g nano-composite, the optimum pH value, the quantity, the concentration and reaction time of glutaraldehyde are 7.0, 10 mL, 10% and 4 h, respectively. In the second step the optimum pH value, reaction temperature and reaction time of laccase are 5.0, 0 ℃ and 2 h, respectively. The average diameter of the immobilized laccase is 78 nm, the saturation magnetization (Ms) of the immobilize laccase is 23.45 A·m2/kg and the surface area of the immobilized laccase is 115.03 m2/g. When 2.0 mg/mL laccase solution is used under optimum conditions and 2, 2′-azino-bis-(3-ethylbenzothiazoling-6-sulphonic acid) is used as substrate, the activity of immobilized laccase reaches 1 430 U/g (nano-composite) and the Michealis-Menten constant(Km) of the immobilized laccase is 2.38×10?5 mol/L.

Interaction of Coal Humic Acids with Fungal Laccase

Humic acids (HA) are one of the main environmental factors controlling the fate and behavior of the compounds released into the environment. In particular, they are universally considered of great importance in determining soil extracellular enzyme activity and stability via association with essential soil enzymes. The objective of this study was to investigate the interaction of coal HA with an extracellular multicopper oxidase laccase (EC 1.10.3.2) that catalyze the oxidation of a wide range of reducing substances in the environment. Using size-exclusion chromatography analysis and monitoring laccase activity, the formation of a stable and an enzymatically active complex between HA and laccase was shown. Basing the data obtained by isoelectric focusing of HA-laccase complex, non-covalent character of laccase association with HA was considered and binding of laccase to HA by weak dispersive forces such as van der Waals, hydrophobic, π-π, CH-π and others was hypothesized.

Immobilization of Agaricus Bisporus Laccase on Ceramic-Chitosan Composite Support and Their Properties:Potential for Oily Wastewater Treatment

Laccase was immobilized on the ceramic-chitosan composite support by using glutaraldehyde as the cross-linking reagent. The immobilization conditions and characterization of the immobilized enzyme were investigated. The immobilization of laccase was successfully realized when 3.0 mL of 1.25 mg/mL of laccase at a pH value of 4.0 reacted with 0.15 g of ceramic-chitosan composite support(CCCS) at 4 ℃ for 24 h. The immobilized enzyme exhibited a maximum activity at pH 3.0. The optimal temperatures for immobilized enzyme were 25 ℃ and 50 ℃. The K_m value of immobilized laccase for ABTS was 66.64 μmol/L at a pH value of 3.0 at 25 ℃. Compared with free laccase, the thermal, operating and storage stability of immobilized laccase was improved after the immobilization.

A review of microbial laccase production and activity toward different biotechnological applications

Laccases are versatile enzymes that belong to the multi-copper oxidase family.This enzyme has several biotechnological applications because of its ablilty to oxidize a wide range of phenolic and non-phenolic substrates.However,their large-scale applicability in bioremediation and water treatment is hindered by high salt content and extreme pH values of the polluted media which also affects the stability,recovery and recycling of laccase.Apart from some bacteria,laccase is abundantly present in several lignin-degrading white-rot fungi viz.Ascomycetes,Deuteromycetes,and Basidiomycetes.Recently,lac-case has been employed in the development of biosensors as a medical diagnostic tool,biofuel cells,and in bioremediation purpose to remove herbicides,pesticides,and some toxic chemicals from the soil.However,most of the enzymes including laccase are normally unstable and susceptible to lose their activity over time.This might be avoided by maintaining the activity and lengthening the enzyme's lifespan through the use of appropriate immobilization procedures.The potential of laccase immobilized biocathodes for dye decolorization in microbial fuel cells has recently been studied.Immobilized laccase nanoparticles have potential uses as biocatalyst in the bioremediation of pollutants.In addition,advanced research considering microbial laccase has been conducted for its heterologous expression along with in silico protein engineering to attain maximum enzyme activity which can be potentially applied in different biotechnological sectors.Patent related to laccase also implied that this enzyme can be used as suitable catalyst for the production of promising anti-cancer drugs and even as a significant ingredient in cosmetics.

地衣芽胞杆菌来源的漆酶BlLac的活性和热稳定性改造

漆酶是一种重要的绿色生物催化剂,在环境修复等领域具有广泛的应用前景。然而,漆酶易受环境因素的影响,比如pH值和温度超过适宜范围,会导致结构的破坏以及活性的快速丧失,从而使漆酶的实际应用受到限制。该文对地衣芽胞杆菌来源的漆酶BILac(GenBank:AAU23292.2)进行了重组表达改造,利用同源建模和分子对接,针对突变底物结合能的优化和二硫键的预设构建了2个突变文库,并实现了突变文库之间的组合突变,最终筛选获得活性和热稳定性均较好提升的突变体C189A-S251C,其对底物ABTS的催化效率达到了野生型的6.3倍,T m值由野生型的54℃提高至57℃,在40℃的半衰期由野生型的2.5 h提高至5.5 h,并且在pH稳定性和最适温度的研究中发现,该突变体提高了对酸性环境的耐受力,最适温度也从37℃提高到了50℃。这一改造将使漆酶BlLac在环境等方面的应用潜力得到提升,同时,该研究所采用的设计策略,为蛋白质改造提供了可行的解决方案。

漆酶:一种新型靶标在农业杀菌剂开发中的潜在应用

目前杀菌剂分子设计多依赖于已知的靶标蛋白,但重复针对相同靶标使用杀菌剂,无疑会增大有害生物对药剂的交互抗性风险。因此,基于新的作用靶标开发新型作用机制的杀菌剂,可以有效解决病原菌对现有杀菌剂的抗性难题。漆酶是二羟基萘黑色素生物合成途径中的关键酶,目前许多研究表明其缺失可使真菌生长发育及致病侵染受到影响,可作为农用杀菌剂潜在靶标进行系统研究。本文介绍了漆酶的结构与功能,并着重介绍了近几年有漆酶抑制活性的化合物作为潜在杀菌剂的研究进展,可为更多新型漆酶抑制剂作为杀菌剂的研究提供指导。

BL Lac天体处于高态与低态时的短时标光变的差异及机制研究

分析了１９９３－１９９５年ＢＬＬａｃ、ＯＪ２８７和ＰＫＳ０７３５＋１７８三颗ＢＬＬａｃ天体光变的监测结果，并结合文献上他人发表的资料，发现一些ＢＬＬａｃ天体处于高态时活动性较强，短时标光变频繁发生，而处于低态时活动性较弱，短时标光变很少发生．文中提出活动星系核的相对论性喷流方向会改变，从而导致长期光变；高态是喷流方向与观测者视线夹角较小的表现，而低态则是夹角较大的表现。利用该模型能够解释ＢＬＬａｃ天体短时标光变行为在高态和低态下的差别，并能很好地解释Ｔａｋａｌｏ等人监测ＯＪ２８７时发现的新现象．

黄孢原毛平革菌漆酶基因lac1680的克隆、表达及产酶研究

以先前筛选到的高产漆酶黄孢原毛平革菌(Phanerochaete chrysosporium)为模板,利用同源克隆技术合成一个全长为1 680 bp的漆酶基因,核苷酸及氨基酸序列比对显示该基因与真菌漆酶基因有较高的同源性,将其命名为lac1680,将该基因连接到构建好的p ET24a载体上,并转入表达菌株BL21 Escherichia coli(DE3)中,经对重组菌预表达的全菌裂解物进行SDS-PAGE检测,获得75 k D目的条带,表明诱导表达成功。随后利用NI-NTA层析柱对洗脱下来的lac1680蛋白进行纯化,纯化回收后,漆酶纯度可达98%以上。通过对比基因工程菌和黄孢原毛平革菌野生菌株不同培养时间产酶活力,结果表明构建好的工程菌活力比原菌酶活力有明显的提高,提高了近39%。

BL Lac天体的光变

大幅、快速光变是BLLac天体的定义特征之一 ,光变研究一直是BLLac天体研究的重要内容。本文对BLLac天体的光变研究成果和BLLac天体的基本理论以及一些相关的其它研究方向的成果作了较为全面的总结 ,指出了目前研究中存在的一些不足或可以进一步发展之处 ,并对其中的几个问题进行了研究。全文共分七章。第一章介绍了光变研究的意义和BLLac天体光变研究的概况 ,着重总结和比较了光变研究的方法。第二章简单介绍了活动星系核的特征、分类等与BLLac天体有关的基本知识 ,着重介绍了活动星系核的能源机制—吸积释能 ,和活动星系核的基本模型。第三章在介绍了BLLac天体的基本性质之后 ,简单介绍了BLLac天体的能谱分布及其主要辐射机制研究的最新进展 ,着重介绍了目前比较公认的BLLac天体的理论模型—相对论喷流模型及其推论—BLLac天体与射电星系的统一模型及其最近的研究动态 ;指出 ,和BLLac天体与其母体星系的统一相比 ,BLLac天体自身的统一对相对论喷流理论而言同样具有重要的理论意义。第四章总结了BLLac天体短时标光变从射电到γ射线的各波段的观测研究成果 ;归纳出了BLLac天体短时标光变的特征并介绍了解释这些特征的理论模型—激波模型 ,指出突然大幅变暗现象虽不如闪耀等突然变亮现象频繁但也是一种普?

BL Lac天体研究简介

本文对ＢＬＬａｃ天体的研究现状，目前天体物理界所热衷的问题：尘埃问题，宁静问题、发射线问题、ＢＬＬａｃ天体与ＯＶＶ／ＨＰＱ关系问题，及处理ＢＬＬａｃ天体的流行方案作一个介绍。

BL Lac天体的光变研究

讨论了BL Lac天体光变的基本特征和BL Lac天体研究的理论模型,并利用吸积盘理论对X射线选 BL Lac天体MrK421的光变特性作了较满意的解释.

特殊BL Lac型天体的VLBI观测研究

BL Lac型天体是一类特殊的活动星系核,根据辐射能谱的不同可以分为低峰值频率BL Lac型天体(LBLs),中间峰值频率BL Lac型天体(IBLs),高峰值频率BL Lac型天体(HBLs),以及极高峰值频率BL Lac型天体(UHBLs)。不同类型BL Lac型天体具有不同的辐射特性,通过射电、光学、X射线、伽玛射线等多波段的观测可以发现很多特殊类型及辐射特性的BL Lac型天体,而甚长干涉测量技术(Very Long Baseline Interferometry,VLBI)是目前分辨率最高的观测技术,也是探测致密核区结构的唯一手段。主要介绍目前对BL Lac型天体的VLBI观测研究概况,以及对其中一些特殊BL Lac型天体VLBI研究的进展及初步结果。

Mixed activation and mixed inhibition of the oxidoreductase activity of Rhus vernicifera laccase by different Pd^(2+) ion concentration

Kinetic experiments were performed to study the effects of Pd2+ ion on the oxidation of 5,6-dibro-mo-2,3-dicyanohydroquinone catalyzed by Rhus vernicifera laccase under condition of pH 4.5 and 30 × 0. 1℃ . The results showed that the mixed activation could be observed when Pd2+ ion was at low concentrations. The competitive and non-competitive activation constants were 9 × 10 and 2 × 10-6 mol/L, respectively. With the increase of Pd2+ ion concentration, the activation was gradually converted into mixed inhibition, and the competitive and non-competitive inhibition constants were 6 × 10-6 and 32 × 10-6 mol/L, respectively.

射电选BL Lac天体(RBLs)的观测性质(Ⅱ)(英文)

收集了ＢＬＬａｃ天体的有关信息作为前文（Ｆａｎ＆Ｌｉｎ１９９６）的补充，并在文中加入了具有喷流、具有超光速子元和具有高能射线辐射的ＢＬＬａｃ天体表。同时在表１ｃ中列出了ＢＬＬａｃ天体的发射线，对偏振度与大幅光变的统计相关在相对论成束框架下给予了解释。

可提高漆酶酶活的载锰活性炭-纤维素复合吸附剂的制备及应用

利用废弃的载锰活性炭与纤维素,通过溶剂再生法制备了一种可用于固定化漆酶、提高固定化漆酶酶活的载锰活性炭-纤维素复合吸附剂,并用其固定化漆酶制备漆酶-载体材料。研究结果表明,Mn^(2+)含量对漆酶-载体材料酶活有一定的影响,复合吸附剂中Mn^(2+)含量为1.5 mg/g时,漆酶-载体材料酶活最高;漆酶固定化的最佳工艺条件为固定化温度45℃、pH值=4,初始酶浓度6.5 g/L;在最佳固定化反应条件下,漆酶-载体材料的漆酶固定化率最高达48.24%;漆酶-载体材料的热稳定性和pH稳定性均得到明显增强;利用漆酶-载体材料降解双酚A,降解率达68%,重复使用5次后的双酚A相对降解率仍可达51%。

具有类漆酶活性的铜基纳米酶检测及降解酚类化合物的研究进展

酚类物质是一类高毒性的有机污染物,对生态安全和人类健康构成严重威胁。因此,检测和去除酚类物质至关重要。天然漆酶作为一种多酚氧化酶,能够高效地催化多酚氧化生成水,具有绿色高效的多功能生物催化剂特性,在生物传感和污染物降解领域具有重要应用价值。然而,天然漆酶的成本高、稳定性差且难以回收利用,这些问题限制了其实际应用。为解决上述问题,研究者开发了纳米酶,这是一种具有类似天然酶催化特性的功能材料,能够弥补天然酶的不足。受天然漆酶活性中心含有4个配位铜原子的启发,设计并合成了铜基纳米酶材料。本文主要介绍了漆酶活性的评价方法,以及由不同有机化合物配体(有机小分子、核苷酸和氨基酸等)组成的铜基有机杂化纳米酶,并探讨了配位原子和配位环境对漆酶活性的影响。同时,总结了铜基有机杂化材料在酚类物质检测和降解方面的应用进展,并展望了具有类漆酶活性的铜基有机杂化材料的发展趋势。

一株白腐真菌固体发酵产酶条件优化

目的:漆酶是一种含铜的多酚氧化酶,能催化多种酚类化合物和芳香族胺氧化物,在食品、造纸、能源、高分子化合物合成等领域有着广阔的应用前景。本研究以一株白腐真菌为研究对象,利用农作物秸杆为原料进行固体发酵,对其产漆酶条件进行优化,为更好利用这一真菌资源提供技术支持。方法:采用单因素实验,从发酵时间、料液比、温度、pH等四个方面对白腐真菌固体发酵产漆酶条件进行优化。结果:在培养基质为玉米芯与麸皮,接种量为10%、发酵时间为4 d、料液比为1∶2.5、发酵温度为28℃、pH 5.5时产漆酶活性最强,漆酶活性达98.99 U/g。结论:该白腐真菌具有较强的利用农作物秸杆产漆酶能力,可为其在农作物秸杆等生物质能源的综合利用提供理论依据。