漆酶改性木质素磺酸钠的结构表征及吸附特征

采用漆酶对木质素磺酸钠(木钠)进行改性, 通过凝胶渗透色谱、 电位滴定、 红外光谱和核磁共振等测试方法研究了漆酶改性木钠的结构特征, 结果表明, 漆酶改性对木钠的磺酸基及表面电荷含量影响很小; 在漆酶对木钠的改性过程中既有解聚作用, 又有聚合作用, 反应初期, 漆酶使木钠发生脱甲基反应及部分链接键的断裂, 使酚羟基含量增大和分子量降低, 随着改性时间的延长, 酚羟基含量减少, 分子量变大, 聚合作用占主导地位. 采用静电逐层自组装技术研究了漆酶改性对木钠吸附特征的影响, 紫外光谱以及原子力显微镜的研究结果表明, 当漆酶改性时间为2 h时, 在平板上的吸附量及吸附膜的表面粗糙度最大, 木钠分子呈扁长的椭球形, 而改性时间为36 h时, 木钠聚合为近似球形的大分子结构, 空间位阻增大, 吸附量及吸附膜的表面粗糙度降低.     

漆酶催化对苯基苯酚的聚合

漆酶(EC 1．10．3．2)是一种氧化还原酶,可应用于催化氧化还原反应和高分子合成领域．聚酚类物质由于具有丌电子共轭结构,通过掺杂可以表现出一定的导电性,而有机导电聚合物是一类有着广泛应用前景的功能材料．此外这类物质还具有较高的三阶非线性系数,是一类重要的有机非线性光学材料,是激光技术的重要物质基础之一．而取代苯酚类物质由于其结构的特殊性,一般很难用化学方法使其聚合．有报道用辣     

Comparison of TEMPO and its derivatives as mediators in laccase catalysed oxidation of alcohols

A series of TEMPO (2,2′,6,6′-tetramethylpiperidinyl-1-oxy) derivatives were studied as mediators of laccase (from Trametes versicolor) in the oxidation of benzyl alcohol and 1-phenylethyl alcohol. TEMPO (1), 4-hydroxy-TEMPO (2) and 4-acetylamino-TEMPO (4) turned out to be the most active mediators for laccase. In addition, 4-acetylamino-TEMPO and 4-hydroxy-TEMPO were more active in the oxidation of 1-phenylethanol compared to TEMPO. For these mediators kinetic isotope effects in the range of 2.1-3.2 were observed for α-monodeutero-p-methylbenzyl alcohol oxidation. These values are consistent with a mechanism involving oxoammonium intermediacy. Competition experiments between benzyl alcohol and 1-phenylethanol showed that TEMPO and its derivatives react faster with primary alcohols than with secondary alcohols, also in line with the proposed mechanism.     

介孔SiO2/Fe3O4中空磁性微球的漆酶固定化

以介孔SiO2/Fe3O4磁性中空微球作为载体,采用物理吸附法对漆酶进行固定化,考察了时间、温度和pH值对漆酶固定化效果的影响,并对固定漆酶的活性及稳定性进行了研究.结果表明,介孔SiO2/Fe3O4磁性中空微球吸附漆酶分子后,介孔材料的比表面积与孔体积均减小.在3 h时复合微球对漆酶的吸附达到平衡,复合微球中介孔SiO2对漆酶的有效固定量为689 mg/g,大大高于纯介孔材料MCM-41的漆酶固定量(319 mg/g).在pH=3～6的条件下,复合微球中固定漆酶仍保持70%以上的相对酶活.当温度不高于60℃时,固定漆酶的相对酶活仍保持65%以上.固定漆酶的pH稳定性和热稳定性都明显优于游离漆酶,固定漆酶的米氏常数为1.05 mmol/L,与游离漆酶相比,固定漆酶与底物的亲和力有所降低.当2,4-二氯苯酚的浓度为10 mg/L时,固定漆酶对其去除率在6 h时达到81.6%,表现出很好的催化活性.     

Ultrasonic pilot-scale reactor for enzymatic bleaching of cotton fabrics

The potential of ultrasound-assisted technology has been demonstrated by several laboratory scale studies. However, their successful industrial scaling-up is still a challenge due to the limited pilot and commercial sonochemical reactors. In this work, a pilot reactor for laccase-hydrogen peroxide cotton bleaching assisted by ultrasound was scaled-up. For this purpose, an existing dyeing machine was transformed and adapted by including piezoelectric ultrasonic devices. Laboratory experiments demonstrated that both low frequency, high power (22 kHz, 2100 W) and high frequency, low power ultrasounds (850 kHz, 400 W) were required to achieve satisfactory results. Standard half (4 g/L H₂O₂ at 90 °C for 60 min) and optical (8 g/L H₂O₂ at 103 °C for 40 min) cotton bleaching processes were used as references. Two sequential stages were established for cotton bleaching: (1) laccase pretreatment assisted by high frequency ultrasound (850 kHz, 400 W) and (2) bleaching using high power ultrasound (22 kHz, 2100 W). When compared with conventional methods, combined laccase-hydrogen peroxide cotton bleaching with ultrasound energy improved the whitening effectiveness. Subsequently, less energy (temperature) and chemicals (hydrogen peroxide) were needed for cotton bleaching thus resulting in costs reduction. This technology allowed the combination of enzyme and hydrogen peroxide treatment in a continuous process. The developed pilot-scale reactor offers an enhancement of the cotton bleaching process with lower environmental impact as well as a better performance of further finishing operations.     

漆酶催化半醌自由基中间体动力学的模拟研究

基于漆酶催化5,6-二溴-2,3-二氰基氢醌生成半醌自由基的动力学过程建立了相应的动力学模型,通过对实际动力学曲线的模拟,建立了半醌自由基的生成与衰减机理,为进一步研究半醌自由基与其它生物活性物质的相互作用奠定了基础.     

Development of Quercetin Biosensor Through Immobilizing Laccase in a Modified β‐Cyclodextrin Matrix Containing Ag Nanoparticles in Ionic Liquid

A biosensor containing Ag nanoparticles in ionic liquid (IL) 1‐butyl‐3‐methylimidazolium bis(trifluoromethylsulfonyl)imide (BMI.Tf₂N) and laccase (Lac) immobilized on β‐cyclodextrin modified with epichlorohydrin (β‐CDEpi) was developed for quercetin determination. Lac catalyses the oxidation of quercetin to quinone, which is then reduced on the biosensor surface and the resulting current was investigated by square‐wave voltammetry. The β‐CDEpi support was analyzed by scanning electron microscopy and Ag‐BMI.Tf₂N by transmission electron microscopy and X‐ray diffraction. The linear range (0.499–7.407 μM) and low detection limit (0.037±0.004 μM) show that the proposed biosensor is suitable for quercetin determination in real samples.     

平菇液体培养液全波长光谱特性与漆酶产量关系研究

漆酶是一种广泛分布在真菌中的酶,其底物有非专一性,在木质素的降解和环境保护方面有重要作用.为测定和估计平菇菌丝液体发酵期间的漆酶产生量,使用紫外-可见分光光度计对菌液进行了全波长扫描,结合常规化学测定方法和平板固体培养基培养时的漆酶圈直径来进行研究,探明粗酶液全波长光谱特性与漆酶产生量的关系.结果发现,发酵粗酶液全波长扫描图谱在300 nm处的波峰数目与漆酶的产量呈现相关关系;300 nm处吸光值大于1.5的波长范围逐渐增大,11天的培养期间其波长范围从5 nm变化到80 nm与漆酶的产量呈正相关关系;在320～400 nm出现有规律的光吸收,且在349,365,388 nm处各出现光吸收峰,其中在365 nm处出现最大吸收,且吸光值均与漆酶的产量呈正相关关系.据此可以应用于粗酶液中漆酶含量的估计或测定.与常规化学分析相比,全波长扫描方法有测定速度快、简单、节省溶剂、无化学污染等优点,为实际工业生产中的漆酶产量估计和测定提供了新方法.     

漆酶在催化合成生物活性化合物中的研究进展

近年来市场对具有营养和药用价值的活性化合物的需求量逐年增加,传统的生产方法已无法满足该类化合物的大规模应用。漆酶是近些年广受欢迎的生物催化剂之一,它可以在温和的条件下催化活性化合物的高效合成,并且有极大潜力取代传统的工业生产方法。本文着重回顾了近十年来漆酶在催化合成活性化合物中的应用,并对漆酶的结构及作用机制进行了介绍;同时指出了漆酶工业化应用中存在的一些问题,比如漆酶产量不足、部分酶促反应介质不适于工业化应用等。通过异源表达、筛选高产菌株提高漆酶产量、使用固定化技术和蛋白质工程提高漆酶的使用寿命、开发更加高效低廉的反应介质系统与寻找新的漆酶底物相结合来降低漆酶的应用成本是今后主要的发展趋势。     

聚芳酰胺-多壁碳纳米管混合物固定漆酶电极的电化学行为

以聚芳酰胺-多壁碳纳米管混合物为载体,利用漆酶表面氨基与聚芳酰胺主链端羧基的共价偶联以及碳纳米管与漆酶间的疏水作用,构筑了具有较高稳定性和电催化活性的漆酶修饰电极.并对该固酶修饰电极的固酶量、酶活力、电化学行为及其电催化氧还原的性能进行了表征.对漆酶分子具有亲和力的聚芳酰胺芳环结构及聚芳酰胺端羧基与漆酶表面氨基的共价偶联避免了漆酶的脱落和变性.而碳纳米管与聚芳酰胺的混合使得该三维修饰电极具有良好的电子导电性,并成功地实现了漆酶的氧化还原活性位与电极之间的直接电荷转移,这一点可由在0.73和0.38V附近观察到漆酶的T1和T2(漆酶的T1,T2铜活性位的形式电位分别为0.78和0.39V(vsNHE))铜活性位的两对氧化还原峰确认.漆酶的担载量为56.0mg·g-1,具有电化学活性的漆酶占总担载漆酶量的68%.在pH=4.4磷酸盐缓冲溶液中,该修饰电极上氧气还原的起始电位为0.55V,其对氧气的米氏常数KM为55.8μmo·lL-1,对氧气的检测限为0.57μmo·lL-1.在4℃下保存两个月后能实现直接电荷转移的漆酶量仅下降了14%左右而氧还原超电势提高了约50mV.结果表明该修饰电极有望用作酶基生物燃料电池的阴极和电流型氧气传感器.