漆酶固定化及其在小型酶反应器中的应用

以尼龙网为载体,戊二醛为交联剂,固定化真菌漆酶。用固定化酶在自行设计的小反应器中降解低浓度造纸废水,试验结果如下：当废水COD的浓度控制在3000 mg/L左右,降解时间为26h,废水的COD去除率达到35%。     

漆酶的固定化及其在生物传感器方面的应用

漆酶是一类含铜的多酚氧化酶,它能够催化许多酚类和非酚类物质的氧化.固定化漆酶能改善漆酶的稳定性,实现酶制剂的重复连续使用,具有重要意义.该文综述了漆酶固定化的各种方法,阐述了漆酶相关活性、机械性能和功能等内容,并对漆酶固定化在生物传感器方面的应用作了介绍.     

壳聚糖载体的改性及其用于固定化漆酶的研究

利用有机酸改性壳聚糖,并用交联法制备酸化的壳聚糖载体,然后用改性壳聚糖载体固定漆酶。甲酸、乙酸改性壳聚糖的最适条件:壳聚糖与甲酸、乙酸的质量摩尔比(g/mol)分别为100∶1、100∶1.5,戊二醛的质量分数为1%,缓冲溶液的pH分别是4.4、5.0,反应时间为3h;壳聚糖与酒石酸、草酸的质量摩尔比(g/mol)分别为100∶0.5、100∶2,戊二醛的质量分数为2%,缓冲溶液的pH分别是3.6、4.2,反应时间为4.5h。不同有机酸改性的壳聚糖用于漆酶的固定,其酶活都有不同程度的提高,尤其用酒石酸改性的壳聚糖载体效果最好,其酶活提高了57%。     

包埋法固定化真菌漆酶及其应用研究

采用海藻酸钠包埋法固定真菌漆酶,海藻酸钠和CaCl2的最佳浓度分别为3%和4%,最佳给酶量为30U,最大回收率为48.0%.与游离漆酶相比,固定化漆酶的热稳定性有明显改善,最适反应pH向酸性方向漂移0.5,最适反应温度提高了5℃.使用固定化酶处理低浓度造纸废水,运行8批次后残留酶活为64%.     

白腐真菌漆酶的固定化及其应用研究*

以尼龙网为载体,戊二醛为交联剂,进行固定化真菌漆酶的条件优化和性质研究,优化条件为：尼龙网在5％的戊二醛溶液中交联6h后,加入30U漆酶溶液固定8h,酶活回收率为50．3％。与游离酶相比,固定化漆酶的热稳定性明显提高,最适pH值略有下降。用该固定化漆酶处理低浓度造纸废水,经过8批次连续试验,酶活保留52％。     

桦褶孔菌漆酶固定化及其对染料的降解

采用壳聚糖交联法和海藻酸钠-壳聚糖包埋交联法固定化桦褶孔菌产生的漆酶,探讨最佳固定化条件,固定化漆酶的温度,pH稳定性及操作稳定性,并以两种固定化酶分别对4种染料进行了降解.结果表明:(1)壳聚糖交联法固定化漆酶的最佳条件为:壳聚糖2.5%,戊二醛7%,交联时间2h,固定化时间5h,给酶量1g壳聚糖小球:1mL酶液(1U/mL),固定化效率56%;(2)海藻酸钠-壳聚糖包埋交联法固定化漆酶的最佳条件为:海藻酸钠浓度4%,壳聚糖浓度0.7%,氯化钙浓度5%,戊二醛浓度0.6%,给酶量4mL 4%海藻酸钠:1mL酶液(1U/mL),固定化效率高达86%;(3)固定化的漆酶相比游离漆酶有更好的温度和pH稳定性;(4)比较两种固定化漆酶,海藻酸钠-壳聚糖包埋交联法固定化酶的温度及酸度稳定性要优于壳聚糖固定化酶,但可重复操作性要弱于后者,两者重复使用8次后的剩余酶活比率分别为71%及64%;(5)两种固定化酶对所选的4种不同结构的合成染料均有较好的降解效果,其中壳聚糖固定化酶对茜素红的降解效果及重复使用性极佳,重复降解40mg/L的茜素红10次,降解率仍保持在100%.     

基于金属有机框架材料酶的固定化策略及其应用

金属有机骨架(MOFs)是由有机配体和金属离子或团簇通过配位键自组装形成的具有分子内孔隙的有机无机杂化材料,由于其比表面积大和化学稳定性、可调控的孔隙以及多样性被越来越多地用于生物分子的装载尤其是酶的固定化领域.本文综述了近年来国内外制备MOFs以及MOFs基的复合材料固定化酶的制备方法、改进策略和应用,并对MOFs基...     

漆酶在磁性壳聚糖微球上的固定及其酶学性质研究

以磁性壳聚糖微球为载体,戊二醛为交联剂,共价结合制备固定化漆酶。探讨了漆酶固定化的影响因素,并对固定化漆酶的性质进行了研究。确定漆酶固定化适宜条件为：50 mg磁性壳聚糖微球,加入10mL 0.8mg/mL 漆酶磷酸盐缓冲液（0.1mol/L,pH 7.0）,在4℃固定2h。固定化酶最适pH为3.0, 最适温度分别为10℃和55℃,均比游离酶降低5℃。在pH 3.0,温度37℃时,固定化酶对ABTS的表观米氏常数为171.1μmol/L。与游离酶相比,该固定化漆酶热稳定性明显提高,并具有良好的操作和存储稳定性。     

固定化真菌漆酶降解氯苯嘧啶醇农药

在葡萄糖培养基中,栓菌420(Trametes sp.420)经6mmol/L邻甲苯胺诱导,漆酶活力达到4535U/L(愈创木酚法)。用弱阴离子交换层析可分离得到纯化漆酶。以壳聚糖为载体,戊二醛为交联剂对漆酶进行固定化,30g壳聚糖与30U漆酶混合,酶活回收率高达69%。在酸性条件下,固定化漆酶对农药氯苯嘧啶醇具有良好的降解作用。     

固定化漆酶对染料酸性紫43的脱色和降解

采用自制复合型载体丙烯酸酯类聚合物固定的漆酶对染料酸性紫43进行脱色处理。研究了固定化酶的用量、底物浓度、温度、pH对其降解效果的影响。结果表明,固定化漆酶脱色降解酸性紫43的适宜条件为:酶用量12.5 U/mL,染料浓度150 mg/L,反应的温度范围在45~55℃,pH值范围在4.5~5.0。在上述条件下降解4h,染料酸性紫43脱色率能达到98.5%。重复分批使用固定化漆酶处理染料酸性紫43,在使用8批次后,脱色率仍然能保持在90%以上,其催化效率得到了很大提高。     

Immobilization of laccase by encapsulation in a sol-gel matrix and its characterization and use for the removal of estrogens

Laccase from Myceliophthora thermophila was immobilized by encapsulation in a sol-gel matrix based on methyltrimethoxysilane and tetramethoxysilane. The amount of laccase used for the preparation of the hydrogel was in the range 2.2-22 mg of protein/mL sol and the corresponding enzymatic activities were in the range 5.5-17.0 U/g biocatalyst. The kinetic parameters of the encapsulated laccase showed that the immobilized enzyme presented lower affinity for the substrate 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonate) (ABTS). However, the stability of laccase was significantly enhanced after immobilization; thus, both pH and thermal stability improved about 10-30% and tolerance to different inactivating agents (NaN(3) , ZnCl(2) , CoCl(2) , CaCl(2) , methanol, and acetone) was 20-40% higher. The reusability of the immobilized laccase was demonstrated in the oxidation of ABTS for several consecutive cycles, preserving 80% of the initial laccase activity after 10 cycles. The feasibility of the immobilized biocatalyst was tested for the continuous elimination of Acid Green 27 dye as a model compound in a packed-bed reactor (PBR). Removals of 70, 58, 57, and 55% were achieved after four consecutive cycles with limited adsorption on the support: only 10-15%. Finally, both batch stirred tank reactor (BSTR) operated in several cycles and PBR, containing the solid biocatalyst were applied for the treatment of a solution containing the endocrine disrupting chemicals (EDCs): estrone (E1), 17β-estradiol (E2), and 17α-ethinylestradiol (EE2). Eliminations of EDCs in the BSTR were higher than 85% and the reusability of the biocatalyst for the degradation of those estrogens was demonstrated. In the continuous operation of the PBR, E1 was degraded by 55% and E2 and EE2 were removed up to 75 and 60%, at steady-state conditions. In addition, a 63% decrease in estrogenic activity was detected.     

Immobilized laccase for decolourization of Reactive Black 5 dyeing effluent

Reactive Black 5 industrial dyeing effluent was decolourized by free and immobilized laccase. The stability of the enzyme (194 h free and 79 h immobilized) depended on the dyeing liquor composition and the chemical structure of the dye. In the decolourization experiments with immobilized laccase, two phenomenons were observed – decolourization due to adsorption on the support (79%) and dye degradation due to the enzyme action (4%). Dyeing in the enzymatically recycled effluent provided consistency of the colour with both bright and dark dyes.     

Purification and properties of laccase from Botrytis cinerea

The partial purification of an extracellular laccase from Botrytis cinerea is described. Specificity of the enzyme, its K_m for a number of substrates and sensitivity to some inhibitors are described. The enzyme is a typical laccase but has an exceptionally low pI and great stability to acid pH. On gel electrophoresis two isoenzymes could be detected.     

Removal of phenols from mixtures by co-immobilized laccase/tyrosinase and Polyclar adsorption

An enzymatic method for removal of phenols from their mixtures was investigated. Phenols in an aqueous solution were removed after a two-step treatment with co-immobilized laccase and tyrosinase and Polyclar (polyvinylpolypyrrolidone). A laccase from Pyricularia oryzae and mushroom tyrosinase were co-immobilized on Mikroperl in a fixed-bed tubular bioreactor by a rapid and simple method. The support immobilized 95% of the total laccase units and 35% of the total tyrosinase units. Different mixtures of phenols were passed through the column with co-immobilized laccase and tyrosinase. This method removed 42–90% of different phenolic substances by a single passage through the bioreactor. The second step employed Polyclar for additional removal of phenolic substances from mixtures. The degree of removal depends on the nature of the phenols. Complete removal was achieved for a-naphthol, 2,4-dichlorophenol, 4-methoxyphenol, b-naphthol, 4-chloro-3-methylphenol and catehin. The operational stability of the immobilized system was 10–90 h depending on the substrate. The biocatalyst was capable of continuous transformation of different phenols in mixtures. Journal of Industrial Microbiology & Biotechnology (2000) 24, 383–388. Received 12 August 1999/ Accepted in revised form 18 February 2000     

灵芝EIM-40漆酶的分离纯化及酶学性质

采用冻干浓缩、（NH4）2S04盐析、HiTrapphenyl（FF）疏水层析和QSepharose FastFlow离子交换层析对灵芝EIM-40发酵液进行分离纯化,获得纯化漆酶,纯化倍数为14．6,回收率为5．3％。SDS-PAGE银染的结果为单一条带,相对分子质量约为6．53×104。以愈创木酚和2,2-联氮-二（3-乙基-苯并噻唑-6-磺酸）二铵盐（ABTS）为催化底物进行酶学性质研究,最适pH分别为4．8和4．5,最适温度分别为55和50℃,2种底物在pH4．0。5．0范围内,温度低于50℃时,酶的稳定性都很好。以愈创木酚为底物,Km=645．0umol／L;以ABTS为底物,Km=22．2txmol／L。Cu2＋对该酶起激活作用,Fe2＋、Ca2＋、Ba2＋则完全抑制酶的活性。     

Sequential reactors for the removal of endocrine disrupting chemicals by laccase immobilized onto fumed silica microparticles

Abstract

The main objective of this study is the evaluation of the capability of laccase from Myceliophthora thermophila immobilized on fumed silica microparticles (fsMP) for the removal of endocrine disrupting chemicals (EDCs) in two enzymatic reactor configurations. This type of support can also be magnetized to allow the straightforward separation of the biocatalyst under a magnetic field. The support exhibited excellent biocompatibility with the enzyme, superior tolerance to pH and temperature as well as improved stability in comparison with the free enzyme, even in the presence of organic solvents and enzyme inhibitors. The technical feasibility of the removal of EDCs by immobilized laccase was assessed in two types of enzymatic reactors operated in sequential mode: a membrane reactor using fsMP-laccase and a reactor with magnetic separation using magnetized fsMP-laccase. The extent of transformation for the target compounds: bisphenol A (BPA) and 17β-estradiol (E2) was high and comparable to free laccase in both systems (up to 80%). The possibility of reusing the immobilized enzyme, especially for magnetized supports, offers an interesting approach in the development of enzyme based processes for the biotransformation of emerging pollutants.     

Construction and direct electrochemistry of orientation controlled laccase electrode

A laccase has multiple redox centres. Chemisorption of laccases on a gold electrode through a polypeptide tag introduced at the protein surface provides an isotropic orientation of laccases on the Au surface, which allows the orientation dependent study of the direct electrochemistry of laccase. In this paper, using genetic engineering technology, two forms of recombinant laccase which has Cys-6×His tag at the N or C terminus were generated. Via the Au-S linkage, the recombinant laccase was assembled orientationally on gold electrode. A direct electron transfer and a bioelectrocatalytic activity toward oxygen reduction were observed on the two orientation controlled laccase electrodes, but their electrochemical behaviors were found to be quite different. The orientation of laccase on the gold electrode affects both the electron transfer pathway and the electron transfer efficiency of O₂ reduction. The present study is helpful not only to the in-depth understanding of the direct electrochemistry of laccase, but also to the development of laccase-based biofuel cells.