漆树漆酶两种同工酶分离纯化与特性研究

应用柱层析和HPLC方法从漆树漆液中分离提纯漆酶的两种同工酶L1和L2 .两者均为低温酶 ,最适的温度分别为 2 0℃和 13℃ ,pH值分别为 6.7和 7.2 ,等电点分别为 8.6和 9.1.通过十二烷基硫酸钠 聚丙烯酰胺凝胶电泳 ,测得它们的分子量分别为 1.2× 10 5和 1.0 5× 10 5.两种酶均不能催化氧化间氨基苯甲酸 ,对 3 氨基酪氨酸等有很高的反应活性     

漆树漆酶催化氧化对苯二胺反应的微量热法研究

在厌氧条件下，０．１ｍｏｌ／Ｌ的磷酸盐缓冲溶液（ｐＨ＝７．４）中，用微量热法测定了２９８．１５Ｋ时漆树漆酶与对苯二胺反应的摩尔反应焓ΔｒＨｍ＝－１６３．３６±０．３６ｋＪ／ｍｏｌ，米氏常数Ｋｍ＝５．５８×１０－３ｍｏｌ／Ｌ和表观一级反应速率常数ｋ１＝８．６３×１０－３ｓ－１，及此条件下用对苯二胺作底物时的漆酶活性值ＥＡ＝０．０４５ＩＵ．还利用阿伦尼乌斯经验公式计算了该反应的表观活化能，并讨论了酶－底复合过程的结合能ΔＧ０的计算．     

聚马来松香乙二醇酯Cu(Ⅱ)配合物固定化漆树漆酶的研究

制备了聚马来松香乙二醇酯(MEEP)Cu2 、Ni2 、Ca2 和Mg2 金属离子配合物,并分别以4种配合物为载体固定化漆树漆酶,初步探讨了反应时间对酶固定化的影响,考察了固定化酶的性质.实验结果表明,在反应温度为25℃条件下,漆树漆酶的最佳固定化时间为16h;MEEPCu2 配合物固定化酶的固定化结果较好,重复使用6次后,酶相对保留活力为55.0%;该固定化酶的最适作用温度为40℃,pH值范围为5.89～9.23,固定化漆树漆酶具有比游离酶更好的热稳定性和更广泛的pH值适用范围.     

A Modular Approach for Interlocking Enzymes in Whatman Paper

A potentially universal approach is presented for enzyme attachment to cellulose that significantly enhances enzyme stability while retaining high activity, and involves no chemical functionalization of cellulose. Bovine serum albumin (BSA) was interlocked in cellulose to form a protein‐friendly surface (named BSA‐Paper), while also providing COOH and NH₂ groups for subsequent attachment of enzymes. The desired enzyme is then mixed with additional BSA and interlocked on BSA‐Paper. The second BSA layer dilutes and crosslinks the enzyme for improved stability. Laccase was tested as a model enzyme for interlocking on BSA‐Paper, and was found to retain over 100 % activity and was 240 times more stable at 25 °C (half life=180 d) than laccase. This new approach was also tested with a few other enzymes with encouraging results, thus providing a potentially universal method for stabilization of enzymes on cellulose with retention of high activities.     

Fuel‐Free Bio‐photoelectrochemical Cells Based on a Water/Oxygen Circulation System with a Ni:FeOOH/BiVO4 Photoanode

A bio‐photoelectrochemical cell (BPEC) based on a fuel‐free self‐circulation water–oxygen–water system was fabricated. It consists of Ni:FeOOH modified n‐type bismuth vanadate (BiVO₄) photoanode and laccase catalyzed biocathode. In this BPEC, irradiation of the photoanode generates photocurrent for photo‐oxidation of water to oxygen, which is reduced to water again at the laccase biocathode. Of note, the by‐products of two electrode reactions could continue to be reacted, which means the H₂O and O₂ molecules are retained in an infinite loop of water–oxygen–water without any sacrificial chemical components. As a result, the assembled fuel‐free BPEC exhibits good performance with an open‐circuit potential of 0.97 V and a maximum power density of 205 μW cm^?2 at 0.44 V. This BPEC based on a self‐circulation system offers a fuel‐free model to enhance multiple energy conversion and application in reality.     

Methomyl Detection by Inhibition of Laccase Using a Carbon Ceramic Biosensor

A sol–gel derived carbon ceramic biosensor was used for methomyl determination in vegetable extract samples based on the immobilization of laccase from Aspergillus oryzae. Esculetin was chosen as the substrate for laccase in order to measure inhibition by this pesticide. The analytical curve was linear for methomyl concentrations of 0.5 to 12.2 µM with a detection limit of 0.2 µM. The lifetime of the proposed biosensor was 60 days and the recovery from vegetable extract samples ranged from 98.0 to 104.2 %. The results using the proposed method are in agreement with those using HPLC at the 95 % confidence level.     

Laccase from Trametes versicolor

The enzyme laccase was produced by the white-rot fungus Trametes versicolor in repeated batches cultures with immobilized mycelium. Two different culture conditions were used. Enzymes produced were evaluated regarding their stability a thigh temperatures (55°C and 65°C) and at alkaline conditions (pH 7.0 and pH 8.0) having in view the application of these enzymes in biobleaching of hardwood Kraft pulp. Biobleaching experiments were divided in two parts, enzymatic prebleaching followed by chemical bleaching. In the enzymatic prebleaching the enzyme laccase was used at two conditions of pH and temperature, whereas the reaction time was fixed at 1h in all pretreatments. In the chemical bleaching the DEDED and DEpDED sequences were used. The enzyme action was evaluated by Kappa number, viscosity, and brightness at the end of bleaching sequences. There were obtained values of Kappa numbers lower than control assays, viscosities compatible with industrial pulps, and brightness higher than controls, when pulps were pretreated for 1 h with laccase at pH 8.0 and 55°C.     

基于固定化电子媒介体的漆酶电极的制备与应用

采用一种新方法引入媒介体,即将媒介体2,2-连氮-双(3-乙基苯并噻唑啉-6-磺酸)(ABTS)固定在多壁碳纳米管(MWCNTs)上,将壳聚糖与漆酶共同修饰在玻碳电极表面,制备了漆酶电极。实验发现,ABTS能够被牢固地吸附在多壁碳纳米管上,且吸附了ABTS的多壁碳纳米管在水中的分散性能大为改善。由于ABTS的存在,该漆酶电极能够更加有效地催化氧气还原,使氧气的还原电位从-0.1 V升至0.6 V。研究了修饰在多壁碳纳米管上的ABTS的电化学行为,并讨论了碳纳米管修饰量、pH值及温度对漆酶电极催化性能的影响。该方法简单,制备的漆酶电极对氧气还原的催化效率高,有望应用于植入型生物燃料电池。     

邻氨基苯酚与漆树漆酶反应的微量热法研究

在０．１ｍｏｌ／Ｌ的磷酸盐缓冲溶液（ｐＨ７．４）中，用微量热法测定了２９８．１５Ｋ时漆树漆酶与邻氨基苯酚反应的摩尔反应焓ΔｒＨｍ＝２５２．２１±３．５９ｋＪ／ｍｏｌ，米氏常数Ｋｍ＝７．７７×１０－３ｍｏｌ／Ｌ和表观一级反应速率常数ｋ１＝７．２３×１０－３ｓ－１，及此条件下邻氨基苯酚作底物时的漆酶活性值ＥＡ＝０．１９０（ＩＵ）。     

A low-cost biofuel cell with pH-dependent power output based on porous carbon as matrix

A glucose/O2 biofuel cell (BFC) possessing a pH-dependent power output was fabricated by taking porous carbon (PC) as the matrix to load glucose oxidase or fungi laccase as the catalysts. The electrolytes in the anode and cathode compartments contain ferrocene monocarboxylic acid and 2,2'-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) diammonium salt as the mediators, respectively. The power of the BFC was enhanced significantly by using PC as the matrix, rather than glassy carbon electrode. Additionally, the power output of the BFC decreases as the pH of the solution increases from 4.0 to 7.0, which provides a simple and efficient method to achieve the required power output. More importantly, the BFC can operate at pH 6.0, and even at pH 7.0, which overcomes the requirement for cathode solutions of pH<5.0 when using fungi laccase as a catalyst. Operation of the BFC at neutral pH may provide a means to power medical devices implanted in physiological systems. The facile and low-cost fabrication of this BFC may enable its development for other applications.