大孔树脂共价固定化漆酶及其对孔雀石绿的脱色研究

以大孔树脂为固定化载体,戊二醛为表面活化剂,采用共价结合法固定漆酶。以ABTS为漆酶底物,通过对固定化漆酶活性的测定,确定大孔树脂固定漆酶的最佳固定条件为：戊二醛浓度=2%、pH=6、活化时间=2 h、固定时间=10 h。在此条件下制备的固定化漆酶能够保持79%的初始活性,获得的固定化漆酶活力能够达到1．94 U·g-1。在酸性条件(pH=3)下,游离漆酶的活性在3 h内丧失71%,而固定化漆酶仅丧失37%,显示出固定化漆酶良好的环境稳定性。固定化漆酶对孔雀石绿染料的脱色率在5h内可达到96%。     

Enhanced oxidation of benzo[a]pyrene by crude enzyme extracts produced during interspecific fungal interaction of Trametes versicolor and Phanerochaete chrysosporium

The effects of interspecific fungal interactions between Trametes versicolor and Phanerochaete chrysosporium on laccase activity and enzymatic oxidation of polycyclic aromatic hydrocarbons (PAHs) were investigated. A deadlock between the two mycelia rather than replacement of one fungus by another was observed on an agar medium. The laccase activity in crude enzyme extracts from interaction zones reached a maximum after a 5-day incubation, which was significantly higher than that from regions of T. versicolor or P. chrysosporium alone. The enhanced induction of laccase activity lasted longer in half nutrition than in normal nutrition. A higher potential to oxidize benzo[a]pyrene by a crude enzyme preparation extracted from the interaction zones was demonstrated. After a 48 hr incubation period, the oxidation of benzo[a]pyrene by crude enzyme extracts from interaction zones reached 26.2%, while only 9.5% of benzo[a]pyrene was oxidized by crude extracts from T. versicolor. The oxidation was promoted by the co-oxidant 2,2'-azinobis-3-ethylbenzthiazoline-6-sulphonate diammonium salt (ABTS). These findings indicate that the application of co-culturing of white-rot fungi in bioremediation is a potential ameliorating technique for the restoration of PAH-contaminated soil.     

典型金属离子对漆酶催化降解溴代阻燃剂四溴双酚A的影响研究

研究了自然环境、垃圾渗滤液及工业废水中广泛存在的金属离子对真菌漆酶（Trametes versicolor）酶活性以及对漆酶催化降解典型溴代阻燃剂四溴双酚A（TBBPA）的影响.金属离子包括Na⁺、K⁺、Zn²⁺、Cd²⁺、Cu²⁺、Hg²⁺、Fe²⁺、Fe³⁺、Pb²⁺、Al³⁺能抑制漆酶酶活性和降低TBBPA的降解转化率,抑制效应随着金属离子浓度的升高而增大;Ca²⁺、Mg²⁺、Mn²⁺则对漆酶活性和TBBPA去除率均没有显著影响.金属离子的抑制作用主要是通过干扰漆酶的电子传递过程产生的.其中,Fe²⁺和Hg²⁺的抑制效应尤为明显,而二者的抑制机制不同,Fe²⁺竞争消耗O₂,Hg²⁺则通过与漆酶结构中氨基酸络合,改变其构型从而抑制其活性.此外,通过加入介体物质2,2-联氮-二（3-乙基-苯并噻唑-6-磺酸）二铵盐（ABTS）、2,2,6,6-四甲基哌啶-氮-氧化物（TEMPO）等物质能够显著提高金属离子存在下漆酶对TBBPA的去除率.     

反胶束体系中漆酶催化愈创木酚的研究

采用4种不同属性类型的表面活性剂(CTAB,Tween-80, AOT以及鼠李糖脂)构建反胶束体系,以木质素模式物愈创木酚为底物,研究了漆酶在反胶束环境中催化愈创木酚的特性,通过对不同反胶束环境中漆酶酶活的对比,确定在4种反胶束体系中,适合酶解反应的最佳表面活性剂浓度和对应的最佳含水率w0.分别为:CTAB浓度20mmol/L,w0=20;Tween 80浓度50mmol/L,w0=25;AOT浓度30mmol/L,w0=10;鼠李糖脂浓度10mmol/L,w0=30.并考察了表面活性剂类型属性对愈创木酚在反胶束体系中酶解行为的影响和底物动力学.结果表明,最佳表面活性剂浓度以及对应的最适含水率条件下,阴离子表面活性剂AOT和鼠李糖脂的反胶束体系中愈创木酚底物的催化效果较好,鼠李糖脂反胶束体系中底物的催化率最高,反应6h后分别比 CTAB、Tween-80以及AOT反胶束体系中的转化率高44.12%、34.6%、29.43%.     

Removal of 17β-estradiol in laccase catalyzed treatment processes

The removal of 17β-estradiol (E2) in laccase catalyzed oxidative coupling processes was systematically studied in this work. We focused on the influence of pH and natural organic matter (NOM) on the performance of the enzymatic treatment processes. It was found that the optimal pH for E2 removal was between 4 and 6. The removal of E2 was slightly inhibited in the presence of NOM. Enzymatic transformation of E2 was second-order in kinetics with first-order to both the concentrations of the enzyme and contaminant. Mass spectrum (MS) analysis suggested that coupling products were formed through radical-radical coupling mechanism. The results of this study demonstrated that laccase catalyzed oxidative coupling process could potentially serve as a treatment strategy to control steroid estrogens.     

两种介体物质在漆酶降解磺胺类抗生素中的作用

通过比较两种介体物质丁香醛(SA)和1-羟基苯并三氮唑(HBT)对漆酶降解5种典型磺胺类抗生素的影响,考察了介体的起始浓度、pH值、反应温度、漆酶起始浓度等因子的影响,得到介体SA对漆酶催化降解磺胺类抗生素的效果更好.在漆酶+SA反应体系中,反应体系pH值为5~6,反应温度为30℃,SA起始浓度为0.5mmol/L,漆酶起始浓度为0.5mg/mL时,漆酶对5种磺胺类抗生素的降解效果最佳,降解去除率在15min时达到75%左右,1h内均达到97%以上. 本研究表明,适当的介体物质可大大提升漆酶对磺胺类抗生素的降解效果.漆酶在介体物质的作用下对抗生素类污染物的生态净化具有良好的应用潜力.     

In vitro degradation of the herbicide atrazine by soil and wood decay fungi controlled through Elisa technique

The interaction between atrazine, a triazine herbicide, and a series of decay fungi was characterized in terms of biodegradation of the herbicide and its influence on fungal growth. The following fungi were studied: thermophilic cellulolytic (Penicillium sp. 13) and noncellulolytic (Humicola lanuginosa sp. 5 and 12) strains isolated from self‐heated plant composts, mesophilic diphenol oxidase producing strain Mycelia sterilia INBI 2–26, white‐rot fungi Cerrena maxima, Coriolopsis fulvocinerea and Coriolus hirsutus. Competitive enzyme immunoassay was elaborated for detection of atrazine in cultural liquid. During agar plate cultivation the growth of Humicola sp. 5 was promoted by atrazine whereas the growth of Humicola sp. 12 and Penicillium sp. 13 was suppressed whereas M. sterilia INBI 2–26 was not affected by the herbicide. Neither atrazine‐accelerated nor atrazine‐depressed thermophilic strains decomposed atrazine during 21‐day cultivation according to ELISA data. In contrast, white‐rot fungi Coriolus hirsutus, Coriolopsis fuhocinerea and Cerrena maxima degraded nearly 50% of the herbicide in 5‐day submerged cultivation and 80–92% of the herbicide up to the 40th day. The soil strain M. sterilia INBI 2–26 decomposed 70% of atrazine in 17‐day cultivation. The degradation level depended of the time of atrazine introduction to the growing media. The relationships between the degree of atrazine decomposition and laccase and Mn‐peroxidase production were shown.     

漆酶催化氧化雌酮的研究

以空气中氧气为氧化剂,用漆酶催化水中的雌酮,分别研究反应体系的PH值、温度、漆酶浓度、底物浓度对雌酮去除率的影响。结果表明：pH值4—5范围内,pH=4．5时,雌酮的去除率最高,40min后可达91％;适宜温度范围为22．5℃～27．5℃,反应的最适温度为25℃,40min后可达93％;适量增加漆酶浓度可提高反应速度和去除效率,但过量的漆酶会造成酶分子对E1的包裹而降低催化效率;漆酶在较宽的底物浓度范围内对雌酮都有催化效果。     

Enzymatic treatment of estrogens and estrogen glucuronide

Natural and synthetic estrogens from sewage treatment systems are suspected to influence the reproductive health of the animals in the rivers.In this article we investigated the enzymatic treatment of three estrogens (estrone,17β-estradiol,and 17α-ethynyletstradiol) by a fungal laccase which oxidize phenolic compounds with dissolved oxygen.The elimination of the estrogenic activities by enzymatic oxidation was demonstrated by medaka vitellogenin assay.In addition,we developed an enzymatic treatment system comprised of β-D-glucuronidase and the laccase for 17β-estradiol 3-(β-D-glucuronide) degradation.The two enzymes eliminated 17β-estradiol 3-(β-D-glucuronide) and the intermediate,17β-estradiol,efficiently.     

焦化废水中降解酚优势菌漆酶活性研究

本实验选取哈依煤气反应器内的活性污泥沉降物作为菌源,以邻甲基酚做为唯一碳源,分离纯化得菌株Enterobacter sp.j11,测定酚浓度、温度、pH值及同酚浓度下的时间变化对于菌株产漆酶活性的影响。结果表明：温度35℃～40℃、pH6.5～7.5、酚800 mg/L条件下菌株生长状态佳,酶活性高;酚浓度一定800 mg/L时,测定了24 h内漆酶随时间的变化,得知菌株8 h后即适应高酚条件。说明该菌株适应能力强、产漆酶稳定。