漆酶的来源与分离纯化技术

漆酶最早是从漆树所产的生漆中发现的,广泛分布于高等植物和真菌,在昆虫和细菌中也有发现。它是一种含铜的多酚氧化酶,既是聚合酶,也是降解酶,参与愈伤组织形成、木质化过程、生漆成膜、氧化酚类、苯胺类等一系列有机化合物。漆酶的去甲基化作用可以使木质素降解,也能够参与自然界中花色的形成。漆酶的应用以分离纯化为基础,主要用到沉淀、透析、色谱、电泳等技术。     

Immobilization of laccase from Myceliophthora thermophila on functionalized silica nanoparticles: Optimization and application in lindane degradation

This work is focused on immobilization of laccase from Myceliophthora thermophila expressed in Aspergillus oryzae(Novozym 51003? laccase) on amino modified fumed nano-silica(AFNS) and the possible use in bioremediation. Hereby, for the first time, factors affecting the immobilization of Novozym 51003? laccase on AFNS were investigated for defining the immobilization mechanism and optimizing the utilization of AFNS as support for laccase immobilization. The highest specific activity(13.1 IU·mg~(-1) proteins) was achieved at offered 160 mg per g of AFNS and for the same offered protein concentration the highest activity immobilization yield, reaching68.3% after the equilibrium time, at optimum pH 5.0, was obtained. Laccase immobilization occurs by adsorption as monolayer enzyme binding in 40 min, following pseudo-first-order kinetics. The possible use of obtained immobilized preparation was investigated in degradation of pesticide lindane. Within 24 h, lindane concentration was reduced to 56.8% of initial concentration and after seven repeated reuses it retained 70% of the original activity.     

Immobilized laccase on magnetic nanoparticles for enhanced lignin model compounds degradation

As a natural aromatic polymer, lignin has great potential but limited industrial application due to its complex chemical structure. Among strategies for lignin conversion, biodegradation has attracted promising interest recently in term of efficiency, selectivity and mild condition. In order to overcome the issues of poor stability and non-reusability of enzyme in the biodegradation of lignin, this work explored a protocol of immobilized laccase on magnetic nanoparticles(MNPs) with rough surfaces for enhanced lignin model compounds degradation.Scanning electron microscope with energy dispersive spectrometer(SEM-EDS), flourier transformation infrared spectroscopy(FTIR) and thermal gravimetric analysis(TGA) were utilized to characterize the immobilization of laccase. The results showed a maximum activity recovery of 64.7% towards laccase when it was incubated with MNPs and glutaraldehyde(GA) with concentrations of 6 mg·ml~(-1) and 7.5 mg·ml~(-1) for 5 h, respectively. The immobilized laccase showed improved thermal stability and pH tolerance compared with free laccase, and remained more than 80% of its initial activity after 20 days of storage at 4 ℃. In addition, about 40% residual activity of the laccase remained after 8 times cycles. Gas chromatography–mass spectrometry(GC–MS) was utilized to characterize the products of lignin model compound degradation and activation, and the efficiency of immobilized laccase was calculated to be 1–5 times that of free laccase. It was proposed that the synergistic effect between MNPs and laccase displays an important role in the enhancement of stability and activity in lignin model compound biodegradation.     

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。     

Surface characterization and direct bioelectrocatalysis of multicopper oxidases

Multicopper oxidases (MCO) have been extensively studied as oxygen reduction catalysts for cathodic reactions in biofuel cells. Theoretically, direct electron transfer between an enzyme and electrode offers optimal energy conversion efficiency providing that the enzyme/electrode interface can be engineered to establish efficient electrical communication. In this study, the direct bioelectrocatalysis of three MCO (Laccase from Trametes versicolor, bilirubin oxidase (BOD) from the fungi Myrothecium verrucaria and ascorbate oxidase (AOx) from Cucurbita sp.) was investigated and compared as oxygen reduction catalysts. Protein film voltammetry and electrochemical characterization of the MCO electrodes showed that DET had been successfully established in all cases. Atomic force microscopy imaging and force measurements indicated that enzyme was immobilized as a monolayer on the electrode surface. Evidence for three clearly separated anodic and cathodic redox events related to the Type 1 (T1) and the trinculear copper centers (T2, T3) of various MCO was observed. The redox potential of the T1 center was strongly modulated by physiological factors including pH, anaerobic and aerobic conditions and the presence of inhibitors.     

Laccase/TEMPO-modified lignin improved soy-protein-based adhesives: Adhesion performance and properties

The aim of this study was to enhance the water resistance of soy protein (SP) adhesives using laccase/TEMPO-modified lignin. Kraft lignin was depolymerized by laccase enzyme in the presence of TEMPO to expand the oxidation reaction of both phenolic and non-phenolic compounds. This simplified process has the distinct advantage of enhancing lignin-protein interaction. Compared with SP adhesives, lignin-protein adhesives showed a stronger elastic modulus, higher thermal stability, and increased wet adhesion performance. Wet shear strength increased by 106% from 0.693 to 1.429 MPa, and partial wood failure was observed after the test. Better performance was also observed in the three-cycle soaking test. At the same time, the stronger interactions between -COO^- and -NH₂ groups of protein and lignin led to a decrease in flowability and spreadability.     

Loss of rutin and antioxidant activity of asparagus juice caused by a pectolytic enzyme preparation from Aspergillus niger

We found that a commercial pectolytic enzyme preparation from Aspergillus niger (pectinase AN) contained laccase activity that decreased rutin content and antioxidant activity of asparagus juice. This research investigated the effects of pH, temperature, and concentration of pectinase AN on pectinase AN’s laccase activity to decrease rutin content and antioxidant activity of asparagus juice. Asparagus juice was incubated with pectinase AN at different pHs (3.2, 4.5 and 5.8), temperatures (25, 37, and 50 °C) and enzyme concentrations (0.1%, 0.5% and 1%). Rutin content and antioxidant activity of samples was determined by HPLC and 2,2′-diphenyl-1-picrylhydrazyl (DPPH) free radical method, respectively. The rate of loss of rutin and antioxidant activity of asparagus juice was smaller at pH 3.2 than at pH 4.5 and pH 5.8, smaller for 0.1% pectinase AN than 0.5% and 1% pectinase AN. The rate of loss of rutin of asparagus juice was greater at 25 °C than at the other two temperatures. Pectinase AN can decrease rutin content and antioxidant activity of asparagus juice at the selected conditions. But rutin content and antioxidant activity of asparagus juice produced using pectinase AN could be less decreased at pH 3.2 and 0.1% of enzyme with less than 2 h of incubation time. This information was helpful for juice industry to produce juices with high antioxidant activity using pectinase AN.     

Dilution strategy to obtain H2 in the 2nd acidogenic phase from enzymatically pre-treated sugarcane bagasse

Sugarcane bagasse (SCB) pre-treated with laccase was used as substrate in two acidogenic phases (1 P and 2 P). In 1 P, the pretreatment conditions were statistically optimized (pH 4.0, 37 °C, 0.4 U of laccase/mL, 24.0 g SCB/L and 120 min of reaction), which allowed obtaining 84% more H₂ (166.8 mL/L) than SCB in natura (90.4 mL/L). In 2 P, the liquid fraction of the 1 P reactors was diluted (90%) and more 108.5 ml H₂/L was obtained. Furthermore, the quantification of enzymes genes responsible for the hydrolysis (Cel) and H₂ production (Hyd) was carried out. An increase in the expression of both genes was observed from the phase of highest H₂ production rates in P1. In 2 P, the 1 P low genic expression was indicative of the autochthonous bacteria activity. The 2nd acidogenic phase strategy proved to be a promising novelty for the use of pre-hydrolyzed substrates with concomitant production of more value-added products.     

Evaluation of hybrid and enzymatic nanofluidic fuel cells using 3D carbon structures

Membraneless nanofluidic fuel cells are devices that utilize fluid flow through nanoporous media which serve as three-dimensional electrodes. In the case of hybrid fuel cells (HFC) an enzymatic and an abiotic catalyst are incorporated on the electrodes. Here we compared two different HFC. In the first one (HFC-1), glucose oxidase- and Pt-based electrodes were used as bioanode and cathode respectively. This cell reached an open circuit voltage (OCV) of 0.55 V and a maximum power density of 5.7 mWcm^?2. In the second one (HFC-2), AuAg- and laccase-based electrodes were used as anode and biocathode respectively. This cell exhibited an OCV of 0.91 V and a maximum power density of 17 mWcm^?2. Finally, enzymatic electrodes were used to develop a high performance biofuel cell (3.2 mWcm^?2) that exhibited high stability over 4 days. These preliminary results indicate that the incorporation of enzymes into the 3D carbon structures is an efficient alternative for miniaturized nanofluidic power sources.     

Effect of laccase and transglutaminase on the textural and water-binding properties of cooked chicken breast meat gels

The effects of laccase and transglutaminase (TG) on the firmness and weight loss of cooked chicken meat homogenate gels were investigated at laboratory scale. The salt, trisodium pyrophosphate and meat contents were also used as variables. Laccase decreased firmness and increased weight loss of phosphate-free, low-meat (65%) and low-salt (1%) gels, although it modified myosin and troponin T and reacted with isolated myofibrils. By applying both low-salt (1%) and low-phosphate (0.17%) amounts, gel firmness decreased and weight loss increased (p<0.05) greatly. A high dosage of TG significantly improved (p<0.05) the strength of phosphate-free, low-meat and low-salt homogenate gels compared to the corresponding no-enzyme controls. TG improved gel firmness of the low-meat homogenate to the level of the homogenate containing 75% meat. Weight loss was increased significantly (p<0.05) in all cases when the high-TG dosage was used. Enzymes were not capable of improving either texture or water-holding capacity in the low-salt–low-phosphate system. The firmness and cooking loss of the chicken meat products containing different amounts of meat, salt and TG were investigated at pilot scale. Under the conditions and dosages used, TG was capable of improving (p<0.05) firmness of the products without a significant reduction in water-holding capacity.