Laccase biosensor using magnetic multiwalled carbon nanotubes and chitosan/silica hybrid membrane modified magnetic carbon paste electrode

A simple and rapid strategy to construct laccase biosensor for determination of catechol was investigated. Magnetic multiwalled carbon nanotubes (MMCNT) which possess excellent capability of electron transfer were prepared by chemical coprecipitation method. Scanning electron microscope (SEM) and vibrating sample magnetometer (VSM) were used to identify its surfacetopography and magnetization, respectively. Laccase was immobilized on the MMCNT modified magnetic carbon paste electrode by the aid of chitosan/silica (CS) hybrid membrane. Using current-time detection method, the biosensor shows a linear response related to the concentration of catechol in the range from 10⁻⁷ to 0.165×10⁻³ mol/L. The corresponding detection limit is 3.34×10⁻⁸ mol/L based on signal-to-noise ratios (S/N) ≥3 under the optimized conditions. In addition, its response current retains 90% of the original after being stored for 45 d. The results indicate that this proposed strategy can be expected to develop other enzyme-based biosensors.     

Preparation of magnetic chitosan nanoparticles and immobilization of laccase

The magnetic chitosan nanoparticles were prepared by reversed-phase suspension method using Span-80 as an emulsifier, glutaraldehyde as cross-linking reagent. And the nanoparticles were characterized by TEM, FT-IR and hysteresis loop. The results show that the nanoparticles are spherical and almost superparamagnetic. The laccase was immobilized on nanoparticles by adsorption and subsequently by cross-linking with glutaraldehyde. The immobilization conditions and characterizations of the immobilized laccase were investigated. The optimal immobilization conditions were as follows: 10 mL of phosphate buffer (0.1 M, pH 7.0) containing 50 mg of magnetic chitosan nanoparticles, 1.0 mg · mL⁻1 of laccase and 1% (v/v) glutaraldehyde, immobilization temperature of 4°C and immobilization time of 4 h. The immobilized laccase exhibited an appreciable catalytic capability (480 units · g⁻¹ support) and had good storage stability and operation stability. The K _m of immobilized and free laccase for ABTS were 140.6 and 31.1 μM in phosphate buffer (0.1 M, pH 3.0) at 37 °C, respectively. The immobilized laccase is a good candidate for the research and development of biosensors based on laccase catalysis. Funded by Key Project of National Science Foundation of China (No. 60537050) and the National Science Foundation of China (No. 60377032)     

Plackett-Burman设计在漆酶发酵培养基主要影响因子筛选中的应用

通过单因素试验和Plackett-Burman设计法,对灵芝菌漆酶发酵培养基主要因子进行了筛选和优化.通过单因素试验得到漆酶发酵培养基组成为：玉米粉20 g/L,葡萄糖10 g/L,麸皮20 g/L,豆粉10 g/L,KH2PO43 g/L和ABTS 0.025 g/L;采用Plackett-Burman试验设计法,从以上6因素中筛选出了对漆酶产量有显著性影响的主效应因素为：玉米粉、麸皮和ABTS,并得到了以漆酶产量为响应值的线性回归方程,为进一步的响应曲面法优化奠定了基础.     

产耐温漆酶菌的筛选及培养基优化

采用愈创木酚CPDA平板法对由广州地区多个不同场所采集的土样进行了菌种分离纯化,筛选出1株产耐温漆酶的菌株Tr0702,通过形态学观察初步判断该菌为木霉属．该菌所产漆酶的最适反应温度为65℃,在70℃下保温60min后残余酶活保留60％以上;发酵培养基经响应面法优化后关键参数如下：可溶性淀粉44．40g／L、吐温（80）9．00g／L、愈创木酚15．04mmol／L,以此优化培养基发酵酶活可达50．15U／mL     

白构菌液体发酵合成漆酶的培养条件研究

提高底物碳源的聚合度和采用有机氮源有利于诱导白构菌 (Flammulinavelutipes)合成漆酶 ,实验结果表明选择淀粉和黄豆粉作为白构菌液体发酵制取漆酶的碳源和氮源有良好的效果。采用振荡培养能够强化白构菌液体发酵体系内的传质作用 ,更适于菌体的生长及漆酶的合成。以1 80r/min速率振荡培养白构菌 1 6d ,漆酶活力可达到 3 0 8IU/mL ,比静置培养提高 83 .3 %。经活化后的白构菌接种并在振荡条件下培养可显著提高漆酶活力 ,1 80r/min振荡培养 6d后漆酶活力可达到 1 749IU/mL。     

A biosensor based on Coriolopsis gallica laccase immobilized on nitrogen-doped multiwalled carbon nanotubes and graphene oxide for polyphenol detection

The use of nanomaterials allows the design of ultrasensitive biosensors with advantages in the detection of organic molecules. Catechol and catechin are molecules that occur naturally in fruits, and their presence in products like dyes and wines affects quality standards. In this study, catechol and catechin were measured at the nanoscale by means of cyclic voltammetry. The oxidation of Coriolopsis gallica laccase immobilized on nitrogen-doped multiwalled carbon nanotubes (Lac/CN_x-MWCNT) and on graphene oxide (Lac/GO) was used to measure the concentrations of catechol and catechin. Nitrogen-doped multiwalled carbon nanotubes (CN_x-MWCNT) were synthesized by spray pyrolysis and characterized by scanning electron microscopy (SEM), transmission electron microscopy (TEM), and x-ray photoelectron spectroscopy (XPS). Covalently bonded hybrids with laccase (Lac/CN_x-MWCNT and Lac/GO) were generated. Catalytic activity of free enzymes determined with syringaldazine yielded 14 584 UmL⁻¹. With Lac/CN_x-MWCNT at concentrations of 6.4 mmol L⁻¹ activity was 9326 U mL⁻¹, while enzyme activity measured with Lac/GO at concentration of 6.4 mmol L⁻¹ was 9 234 U mL⁻¹. The Lac/CN_x-MWCNT hybrid showed higher stability than Lac/GO at different ethyl alcohol concentrations. The Lac/CN_x-MWCNT hybrid can measure concentrations, not previously reported, as low as 1 × 10⁻⁸ mol L⁻¹ by measuring the electric current responses.     

Immobilization of Pycnoporus sanguineus laccase by metal affinity adsorption on magnetic chelator particles

BACKGROUND: Immobilized enzymes provide many advantages over free enzymes including repeated or continuous reuse, easy separation of the product from reaction media, easy recovery of the enzyme, and improvement in enzyme stability. In order to improve catalytic activity of laccase and increase its industrial application, there is great interest in developing novel technologies on laccase immobilization. RESULTS: Magnetic Cu²⁺‐chelated particles, prepared by cerium‐initiated graft polymerization of tentacle‐type polymer chains with iminodiacetic acid (IDA) as chelating ligand, were employed for Pycnoporus sanguineus laccase immobilization. The particles showed an obvious high adsorption capacity of laccase (94.1 mg g^?1 support) with an activity recovery of 68.0% after immobilization. The laccase exhibited improved stability in reaction conditions over a broad temperature range between 45 °C and 70 °C and an optimal pH value of 3.0 after being adsorbed on the magnetic metal‐chelated particles. The value of the Michaelis constant (K_m) of the immobilized laccase (1.597 mmol L^?1) was higher than that of the free one (0.761 mmol L^?1), whereas the maximum velocity (V_max) was lower for the adsorbed laccase. Storage stability and temperature endurance of the immobilized laccase were found to increase greatly, and the immobilized laccase retained 87.8% of its initial activity after 10 successive batch reactions. CONCLUSION: The immobilized laccase not only can be operated magnetically, but also exhibits remarkably improved catalytic capacity and stability properties for various parameters, such as pH, temperature, reuse, and storage time, which can provide economic advantages for large‐scale biotechnological applications of laccase. Copyright © 2007 Society of Chemical Industry     

白腐菌Coriolus versicolor的培养及产漆酶条件的研究

从液体和固体两种培养体系出发,探索不同培养条件对白腐菌分泌漆酶的影响。液体培养体系中,白腐菌的最佳生长和漆酶分泌条件是缓冲体系起始pH值为5.8、摇瓶转速为180r/min、装液量为100mL/500mL;在所有测试的碳源中,葡萄糖为最佳碳源,在二糖中以蔗糖为碳源,漆酶酶活相对较高,而多糖(如淀粉)不利于漆酶的生成。在所有测试的氮源中,硝酸铵和酒石酸铵组合为最佳氮源,氯化铵和尿素的效果次之,蛋白胨的效果最差。当碳源与氮源质量比(C/N)大于10∶1,即高碳低氮的培养条件有利于漆酶的生成。芳香化合物诱导剂ABTS浓度为0.5～1mmol/L时,对产漆酶的影响最明显,与限氮培养基中产酶相比可提高酶活5倍,约为230IU/mL;0.5mmol/L愈创木酚、1mmol/L阿魏酸和0.05mmol/L二甲基苯胺的添加可提高酶活约2～2.5倍;0.01mmol/L黎芦醇的添加可提高漆酶酶活约1.6倍。由于酪氨酸具有酚类化合物的特点,也可作为一种无毒的天然漆酶诱导剂。菌株漆酶活力在固体培养体系明显低于在液体培养体系。