SBA-15固定化漆酶及其稳定性研究

以大孔径介孔分子筛SBA-15为载体,用吸附法对漆酶进行固定化,以2,2-联氮-二(3-乙基-苯并噻唑-6-磺酸)二铵盐为底物考察了漆酶的最适pH值以及固定化体系中给酶量对固定化漆酶活性及其稳定性的影响。结果表明,在pH值为3时,漆酶具有较好的活性;固定化漆酶的活性随着酶和载体质量比的升高先升高后降低,当酶和载体质量比为48 mg/g时,固定化漆酶的活性最高;与游离漆酶相比,固定化漆酶可以重复利用,经过8批次的反应后,固定化漆酶的剩余活性仍然达到85%以上,表现出良好的操作稳定性。     

固定化漆酶的最佳条件及其稳定性研究进展

介绍了固定化漆酶的最佳条件及其稳定性的研究现状。从吸附法、包埋法、结合法和交联法等四种固定化方法阐述了固定化最佳条件的选择及其原因,并分析了固定化漆酶的操作稳定性、热稳定性和储存稳定性。另外对新型固定化技术和固定化漆酶的稳定性研究前景做了分析和展望。     

漆树漆酶催化单宁酸的氧化反应研究

用分光光度法考察漆树漆酶催化单宁酸的氧化情况,探讨了pH值、反应温度、单宁酸浓度、有机酸和金属离子等对催化反应的影响.结果表明,当反应温度为30℃时,漆树漆酶催化反应效果较好;在pH值为4.2～ 9.8的范围内,反应速率随pH值增大而加快;单宁酸浓度在1.47×10-5 ～ 2.94×10-4 mol·L-1时,反应速率随底物浓度的增大呈线性增加;Cu2 、Mg2 和Ni2 三种金属离子对催化反应有激活作用,其中Cu2 影响最大;而柠檬酸和乙二胺四乙酸对酶催化反应均有抑制作用.     

漆酶催化偶氮染料酸性橙脱色的研究

利用米曲霉菌漆酶对偶氮染料酸性橙进行脱色实验,考察反应时间、pH、加酶量、温度、染料质量浓度等因素对脱色率的影响。结果表明,漆酶催化酸性橙的适宜条件为:反应时间为50 min、pH=6、加酶量为2 mL、反应温度为50℃、染料质量浓度为0. 1 g/L,最适条件下酸性橙的脱色率达88. 32%。同时研究了金属离子、酶抑制剂对酸性橙脱色的影响,结果发现十二烷基硫酸钠(SDS)显著抑制漆酶的催化活性,Fe²⁺对漆酶脱色有完全抑制作用; Mg²⁺对脱色有一定的促进作用。     

漆树漆酶催化甘蔗渣纸浆漂白的研究

利用漆树漆酶在无介体存在条件下对甘蔗渣纸浆进行生物漂白,初步探索了反应时间、漆树漆酶用量、温度及酸度对甘蔗渣纸浆白度的影响。结果表明,在浆浓为3%,漆树漆酶用量1.6 mL/g(对干浆),漂白时间20 min,反应温度20~50℃,pH值为6.1~8.1时,甘蔗渣纸浆白度可提高4%~5%ISO。     

漆树漆酶和真菌漆酶的异同研究

本文在文献的基础上 ,比较了漆树漆酶和真菌漆酶在结构、催化活性、氧化还原电势、等电点、反应动力学参数、分离纯化方法、生物学功能等方面的异同 ,并对此进行了解释 ;最后对漆酶的基础研究和应用研究进行了展望     

杂色云芝漆酶的分离、纯化和酶学特性研究

杂色云芝发酵液,经超滤、DEAE-Sephadex A250柱层析两步分离、纯化出两型漆酶。以愈创木酚与酶带在适当条件下反应呈棕红色谱带鉴定出漆酶谱带。两型漆酶分别命名为LacA和LacB。经测定两漆酶亚基的分子量分别为66.0kDa和30.7kDa;等电点pI分别为4.79和5.18;LacA的最适作用温度是40℃,LacB为50℃;保温2h, LacA的半失活温度为60℃,LacB为90℃;LacA和LacB催化愈创木酚的酶活最适pH值均为4.0; 二者的稳定pH值也都在2.5~5.5之间。LacA催化氧化愈创木酚的Km和Vmax分别为286.5molL-1和292.4nmol mL-1 min-1;LacB催化氧化愈创木酚的Km和Vmax分别为813.0mol L-1和127.4nmol mL-1 min-1。     

漆酶催化聚合对氨基苯酚

漆酶在pH 5.0的醋酸盐缓冲溶液中催化聚合4-氨基苯酚,微型反应器中40℃持续反应10h后得到黑褐色粉末。通过UV-Vis、FT-IR、1HNMR、MALDI-TOF MS、TGA和DSC对产物的结构和性能进行表征。结果表明：所得聚合物在氯仿中不溶,在丙酮、四氢呋喃、N,N-二甲基甲酰胺和二甲基亚砜中均有良好的溶解性能。根据UV-Vis、FT-IR和1HNMR分析得知聚合物中酚羟基保留,氨基参与反应,苯环上的氢部分被取代,并且推测了可能的聚合物结构。根据MALDI-TOF MS、TGA和DSC结果分析,聚合物为低聚物,相对分子质量分布在300Da到1500Da之间,聚合度从3到14。产物的分解温度为223℃,具有良好的耐热性能。     

漆酶的结构与催化反应机理

漆酶是一种广泛分布的多酚氧化酶,主要由单一多肽、铜离子活性中心、糖配基组成。多肽链一般含有18种氨基酸,构成漆酶的结构主体;糖配基因漆酶的来源不同而异,是漆酶多样性的标志之一。漆酶活性中心的铜离子依据光学和磁学特性被分为3种类型:Ⅰ型或蓝型铜,Ⅱ型或通常型铜,Ⅲ型或偶联的双核型铜。漆酶的催化反应机理主要包括酶对底物的电子提取、电子的传递和氧分子对酶的还原三步,是一个通过电子转移发生氧化还原反应的过程。     

漆酶的固定化及其在废水处理中的应用

漆酶是一类多酚氧化酶,它能够催化空气中的氧气直接氧化各种酚类、芳胺等芳香类化合物。固定化漆酶能够延长漆酶的使用寿命、提高漆酶的稳定性及其对环境的耐受性,因此它具有比游离漆酶更好的使用性质,从而在制备生物传感器、治理工业废水等方面有着较广泛的应用。综述了近几年来固定漆酶的主要方法及其在废水处理中的应用,并对固定化漆酶的工业应用前景进行了展望。     

Enhancing laccase stability and activity for dyes decolorization using ZIF-8@MWCNT nanocomposite

The continuous use of chemical dyes in various industries, and their discharge into industrial effluents, results in severe problems to human life and water pollution. Laccases have the ability to decolorize dyes and toxic chemicals in industrial effluents as green biocatalysts. Their possible industrial applications have been limited by poor reusability, low stability, and loss of free laccase action. In this research, laccase was immobilized on zeolitic imidazolate framework coated multi-walled carbon nanotubes (Laccase@ZIF-8@MWCNTs) via metal affinity adsorption to develop an easy separable and stable enzyme. The optimum reaction conditions for immobilized laccase are at a pH of 3.0 and a temperature of 60?℃. The immobilized laccase was enhanced in storage and thermal stability. The results indicated that Laccase@ZIF-8@MWCNTs still maintained 68% of its original activity after 10 times of repeated use. Most importantly, the biocatalytic system was applied for decolorization of different dyes (20?mg·L^?1) without a mediator, and up to 97.4% for Eriochrome black T and 95.6% Acid red 88 was achieved in 25 min. Biocatalysts with these properties may be used in a variety of environmental and industrial applications.     

Cu~(2+)修饰低固载量固定化漆酶的活性研究

由于漆酶这种生物蛋白酶有容易变性失活且不可重复使用的特性,限制了漆酶的应用和发展。因此如何提高漆酶的活性,稳定性以及重复使用性尤为重要。本文采用交叉偶联技术,将漆酶固定在具有核壳结构的磁性纳米粒子Fe3O4/SiO2上,并研究了用Cu2+修饰后的低固载量固定化漆酶的活性变化以及修饰后固定化漆酶的稳定性。实验结果显示:Cu2+修饰后的低固载量固定化漆酶的相对活性提高了54%;而且在温度不太高的情况下,修饰后的低固载量固定化漆酶的相对活性优于修饰前的固定化漆酶;修饰后的固定化漆酶降解浓度为10mg·L-1的普施安染料,重复利用13次后降解率仍然可以达到92%。     

Effect of HBT on the stability of laccase during the decolourization of textile wastewaters

BACKGROUND: This study concerns the assessment of laccase stability during textile industry effluent treatment by laccase in the presence of a mediator RESULTS: The effects of 1‐hydroxybenzotriazole (HBT) and textile wastewater on the stability of a crude laccase prepared from cultures of Trametes trogii during the process of textile industry effluent decolourization were determined. HBT was essential for decolourization of effluent by laccase. Decolourization efficiency decreased when effluent was repeatedly added to the reaction mixture, suggesting inhibition of laccase during the process. Stability of the laccase was not affected by different concentrations of effluent, but inactivation was proportional to HBT concentration. CONCLUSION: Experimental results showed that a redox mediator such as HBT was required for high decolourization rate of textile industry effluent; however free radicals generated from this compound inactivated the laccase used in the process. Copyright © 2009 Society of Chemical Industry     

葡聚糖的环氧化修饰与表征

以1,4-丁二醇二缩水甘油醚（BDE）为环氧化试剂,对葡聚糖（Dex）进行功能化修饰,探讨了投料比、pH值、反应时间等条件对环氧取代度的影响。通过核磁共振氢谱（1HNMR）表征产物的结构,采用多角激光光散射与尺寸排阻色谱联用分析系统（MALLS-SEC）考察产物的分子量及其分布。采用Axen方法测定产物的环氧取代度,并通过检测产物中环氧量随时间的变化情况考察了产物的稳定性。结果表明。环氧基团被成功地引入到葡聚糖的单元节上。并且通过改变反应条件可以对环氧取代度进行调控,得到稳定的环氧化葡聚糖产物;在反应温度为37℃、投料比n（Dex）：n（BDE）=1;30、pH=12．6、反应时间为12h的条件下,环氧取代度高达16％,且4℃的低温下环氧化葡聚糖在水中的稳定性良好。     

酶的寡聚结构与催化稳定性

酶的结构与催化稳定性是生物催化与转化过程中的研究热点之一。与单体酶相比,寡聚酶在进化过程中亚基之间的聚合使其在结构和功能上具有一定的优越性,然而寡聚酶独特的四级结构导致其在制备和应用中存在诸多问题,如制备效率低、催化位点利用率低、催化稳定性差等,其中亚基解离导致的催化稳定性问题在很大程度上限制了其工业化应用。目前,介质工程、多亚基固定化、亚基界面工程和融合蛋白策略被应用于寡聚酶的催化稳定性改造,而寡聚酶至单体酶的改造策略则试图从根本上解决寡聚酶的制备和应用问题,具有较好的应用前景。本文介绍了酶的寡聚结构演替所产生的新功能,总结了寡聚酶在制备和应用中存在的问题,重点阐述了提高寡聚酶制备效率和催化稳定性的策略。     

Immobilization of laccase for oxidative coupling of trans-resveratrol and its derivatives

Trametes villosa Laccase (TVL) was immobilized through physical adsorption on SBA-15 mesoporous silica and the immobilized TVL was used in the oxidative coupling of trans-resveratrol. Higher loading and activity of the immobilized enzyme on SBA-15 were obtained when compared with the free enzyme. The effects of reaction conditions, such as buffer type, pH, temperature and substrate concentration were investigated, and the optimum conditions were screened and resulted in enzyme activity of up to 10.3 μmol/g·h. Furthermore, the oxidative couplings of the derivatives of trans-resveratrol were also catalyzed by immobilized TVL. The immobilized TVL was recyclable and could maintain 78% of its initial activity after reusing it four times.     

云芝漆酶的培养和分离纯化的研究

研究了云芝009菌株漆酶的培养及分高纯化,其适宜的产酶培养基为Dodson低碳培养基,起始pH值为3.5.同时添加黎芦醇对漆产酶具有强烈的诱导作用．高浓度的Cu2+对产本酶也有促进作用,经过超滤,DEAE－Cellulose和SephadexG－100分离纯化,发现云芝009菌株漆酶由组分A和B组成,其中,B组分占总酶活的85％以上。     

有机相温敏性脂肪酶催化剂的结构和催化特性

采用不同链长PEO嵌段的Pluronic分子P123、P105、F127及F108,将其端羟基氧化为醛基,使其与脂肪酶CALB的氨基反应形成纳米结合物。TEM分析结果显示CALB-Pluronic结合物在甲苯中呈现直径为20~40 nm的纳米颗粒。所形成的CALB-Pluronic结合物在4-甲基-2-戊酮中呈现温度响应性,降低温度可使酶催化剂沉淀出来,结合物中的Pluronic高分子PEO嵌段越长,其最低临界共溶温度(LCST)越高。采用甲苯为溶剂、正丁醇及正己酸为底物,37℃下的催化实验结果显示CALB-Pluronic纳米结合物比天然CALB的表观活性提高4~6倍,而反应结束后可通过降温回收酶催化剂,显示出脂肪酶-Pluronic纳米结合物在有机相酶催化中的良好应用前景。     

Decolorisation of synthetic dyes by crude laccase from Rigidoporus lignosus W1

BACKGROUND: Effluents from the dyeing process containing dyes are difficult to degrade biologically. Therefore enzymatic dye decolorisation has received considerable attention recently. In this study the dye decolorisation potential of crude laccase from the white rot fungus Rigidoporus lignosus W1 was demonstrated on an anthraquinone dye, Remazol Brilliant Blue R (RBBR), and a triphenylmethane dye, malachite green (MG). Effects of pH, temperature and ionic strength on laccase activity and decolorisation efficiency were investigated. RESULTS: Native polyacrylamide gel electrophoresis (PAGE) and sodium dodecyl sulfate PAGE confirmed the decolorisation ability of the crude enzyme on RBBR and MG. A single laccase with a molecular mass of about 40 kDa appeared to be involved in the process. Efficient enzymatic decolorisation without redox mediator was achieved, with 39 mg L^?1 MG being removed within 2 h and 160 mg L^?1 RBBR within 1 h at 60 °C and pH 4.5. Although the laccase activity was inhibited in the presence of NaCl, it was renatured gradually in low concentrations of NaCl (<0.8 mol L^?1), resulting in unusual dye decolorisation kinetics. Surprisingly, unusual storage stability at alkaline pH was observed, with the laccase activity being enhanced 1.5–2‐fold after 3 h of incubation. CONCLUSION: Crude laccase with unusual storage stability from the fungus R. lignosus W1 showed excellent decolorisation ability on RBBR and MG without redox mediator. This laccase would seem to be a good candidate for application in dye decolorisation and textile effluent biotreatment. Copyright © 2008 Society of Chemical Industry