Immobilized Laccase on a New Cryogel Carrier and Kinetics of Two Anthraquinone Derivatives Oxidation

A coordinatively immobilized laccase was prepared using a new cryogel type carrier. The support has a wide-pore texture facilitating diffusion of different substrates to the enzyme reaction center. The biocatalyst proved to be efficient in decolorization of two anthraquinone derivatives, namely Acid Blue 62 and bromaminic acid. After 24 h over 80% of the two substrates have been oxidated. The kinetic data (K (m) and V (max)) for the oxidation of the two anthraquinone derivatives, with the free and immobilized enzyme, have been determined and compared. Other parameters, like k (cat) and the specificity constant have been calculated and analyzed. The influence of substrate properties (hydrophobicity, polarity, etc.) has been discussed.     

A New Metal-Chelated Cryogel for Reversible Immobilization of Urease

Poly(2-hydroxyethyl methacrylate-co-glycidyl methacrylate) [poly(HEMA-GMA)] cryogel was synthesized by cryopolymerization technique at frozen temperature. Iminodiacetic acid (IDA) was then attached covalently to the cryogel as a chelating agent. Then, poly(HEMA-GMA)-IDA cryogel was chelated with Ni(II) ions and this novel metal affinity support was used for adsorption of urease from its aqueous solution. Urease adsorption experiments were carried out in a continuous system by using a peristaltic pump. Maximum urease adsorption onto poly(HEMA-GMA)-IDA-Ni(II) cryogel was found to be 11.30 mg/g cryogel at pH 5.0 acetate buffer and in 25 °C medium temperature. Urease adsorption capacity decreased with increasing ionic strength and increasing chromatographic flow rate. Adsorption kinetics of urease onto poly(HEMA-GMA)-IDA-Ni(II) cryogel was also investigated and it was found that Langmuir adsorption model is applicable for this adsorption study. This novel immobilized metal affinity chromatography support was used 10 times without any decrease at their adsorption capacity. It was also observed that urease enzyme was repeatedly adsorbed and desorbed without significant lost in enzymatic activity.     

Immobilization of cellulase in nanofibrous PVA membranes by electrospinning

Electrospinning is a nanofiber-forming process by which either polymer solution or melt is charged to high voltages. With high specific surface area and porous structure, electrospun fibrous membranes are excellent candidates for immobilization of enzymes. In this paper, immobilization of cellulase in nanofibrous poly(vinyl alcohol) (PVA) membranes was studied by electrospinning. PVA and cellulase were dissolved together in an acetic acid buffer (pH 4.6) and electrospun into nanofibers with diameter of around 200 nm. The nanofibrous membranes were crosslinked by glutaraldehyde vapor and examined catalytic efficiency for biotransformations. The activity of immobilized cellulase in PVA nanofibers was over 65% of that of the free enzyme. Nanofibers were superior to casting films from the same solution for immobilization of cellulase. The activity of immobilized cellulase descended with ascending in enzyme loading efficiency and crosslinking time, which retained 36% its initial activity after six cycles of reuse.     

酶固定化技术用载体材料的研究进展

酶的固定化是生物技术中最为活跃的研究领域之一.作为固定化酶技术的重要组成部分,载体的结构及性能在很大程度上直接影响着所得固定化酶的催化活性及操作稳定性.综合性能优良的载体材料的设计与制备是固定化酶技术领域的一个非常重要的研究内容.通过对传统材料的改性和新型载体材料的研究开发,必将促进固定化酶在各个领域的广泛应用.本文综述了近年来国内外有关固定化酶载体材料的研究现状和发展趋势.     

HEMA与HEA辐射共聚制备固定化微生物载体的结构与性能

应用低温辐射技术辐射诱导甲基丙烯酸β-羟乙酯(HEMA)和丙烯酸羟乙酯(HEA)共聚合制备了高分子载体,用增殖细胞技术固定氨氧化细菌。利用红外光谱(FT-IR)、X射线光电子能谱(XPS)、X射线衍射(XRD)、扫描电子显微镜(SEM)以及接触角和含水率的测试对聚合物载体进行了性能表征。结果表明:经充分溶胀后的聚合物表面水接触角几乎为0,含水率为450%,润湿性能良好;聚合物表面具有极性官能团;聚合物的非晶结构有利于小分子尤其是水分子的渗透和扩散,多孔结构有利于微生物的生长和繁殖。以聚合物为载体固定化氨氧化细菌在处理含氨废水的过程中实现了短程硝化,在3种氨氮负荷(100、1502、00 mg/L)条件下,氨氮去除率和亚硝化率可分别达到95%和90%以上。     

Synthesis of New Potential Hydrophobic Agents Composed of Three-Module Type Molecules

A new synthetic route was developed to three-module type potential hydrophobic agents, with the molecule consisting of an N-[3-(triethoxysilyl)propyl]amide anchor part (I), a connecting unit formed upon 1,3-propansultone ring cleavage (II), and a polyfluoroheptyloxy functional hydrophobic spacer (III). Proceeding from commercially available polyfluorinated heptanols 1a and 1b and 1,3-propanesultone 3, potassium sulfonates 4a and 4b were prepared. The reaction of 4a and 4b with phosphorus oxychloride resulted in the first synthesis of fluorine-containing sulfonyl chlorides 5a and 5b, which were reacted with 3-aminopropyltriethoxysilane 6 to give the target N-[3-(triethoxysilyl)propyl]-3-(polyfluoroheptyloxy)propane-1-sulfonamides 7a and 7b. The structures of the compounds were proved by NMR spectroscopy, mass spectrometry, and elemental analysis. The studies of their hydrophobizing properties are in progress.     

Banana Peel: A Potential Substrate for Laccase Production by <Emphasis Type="Italic">Aspergillus fumigatus</Emphasis> VkJ2.4.5 in Solid-State Fermentation

In solid-state fermentation, among various solid supports evaluated, banana peel was found to be an ideal support and resulted into higher levels of laccase (6281.4 ± 63.60 U l⁻¹) along with notable levels of manganese peroxidase production (1339.0 ± 131.23 U l⁻¹) by Aspergillus fumigatus VkJ2.4.5. Maximum levels of laccase was achieved under derived conditions consisting of 80% of moisture level, 6 days of incubation period, 6% inoculum level, and an aeration level of 2.5 l min⁻¹. A column-tray bioreactor was designed to scale up and economize the enzyme production in three successive cycles of fermentation using the same fungal biomass. Thermal and pH stability profiles revealed that enzyme was stable up to 50°C and at varying pH range from 5–9 for up to 2 h. The apparent molecular weight of laccase was found to be 34 ± 1 kDa. MALDI-TOF/TOF analysis of the protein showed significant homology with maximum identity of 67% to other laccases reported in database.     

Magnetic Polyethyleneimine Nanoparticles Fabricated via Ionic Liquid as Bridging Agents for Laccase Immobilization and Its Application in Phenolic Pollutants Removal

In this study, polyethyleneimine was combined with magnetic Fe₃O₄ nanoparticles through the bridging of carboxyl-functionalized ionic liquid, and laccase was loaded onto the carrier by Cu²⁺ chelation to achieve laccase immobilization (MCIL–PEI–Cu–lac). The carrier was characterized by Fourier transform infrared spectroscopy, scanning electron microscope, thermogravimetric analysis, X-ray diffraction analysis, magnetic hysteresis loop and so on. MCIL–PEI–Cu–lac has good immobilization ability; its loading and activity retention could reach 52.19 mg/g and 91.65%, respectively. Compared with free laccase, its thermal stability and storage stability have been significantly improved, as well. After 6 h of storage at 60 °C, 51.45% of the laccase activity could still be retained, and 81.13% of the laccase activity remained after 1 month of storage at 3 °C. In the pollutants removal test, the removal rate of 2,4-dichlorophenol (10 mg/L) by MCIL–PEI–Cu–lac could reach 100% within 10 h, and the removal efficiency could still be maintained 60.21% after repeated use for 8 times. In addition, MCIL–PEI–Cu–lac also has a good removal effect on other phenolic pollutants (such as bisphenol A, phenol, 4-chlorophenol, etc.). Research results indicated that an efficient strategy for laccase immobilization to biodegrade phenolic pollutants was developed.     

Potential Involvement of Both Type I and Type II Mechanisms in M13 Virus Inactivation by Methylene Blue Photosensitization

We have investigated the mechanism of virus photoinactivation with methylene blue (MB) by conducting deuterium oxide (D₂O), azide ion (N₃^-) and oxygen-dependent, studies. Inactivation of M13 bacteriophage and singlet oxygen (¹O₂) generation by MB photosensitization were irradiation dose dependent. Inactivation of M13 was enhanced by D₂O and inhibited by N₃^-, suggesting that ¹O₂ participates in M13 inactivation by MB photosensitization. However, N₃^- did not inhibit M13 inactivation completely. On the other hand, deoxygenating the reaction solution still caused 52-67% of M13 inactivation observed in the presence of oxygen. These results suggest that ¹0₂-mediated (Type II) and sensitizer-mediated (Type I) reactions may both play roles in M13 inactivation by MB photosensitization.     

A New <Emphasis Type="Italic">Stenotrophomonas maltophilia</Emphasis> Strain Producing Laccase. Use in Decolorization of Synthetics Dyes

Laccase activity was detected in a soil bacterium Stenotrophomonas maltophilia AAP56 identified by biochemical and molecular methods. It was produced in cells at the stationary growth phase in Luria Bertani (LB) medium added by 0.4 mM copper sulfate. The addition of CuSO₄ in culture medium improved production of laccase activity. However, one laccase enzyme was detected by native polyacrylamide gel electrophoresis. The enzyme showed syringaldazine (K _m = 53 μM), 2,2’-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (K _m = 700 μM), and pyrocatechol (K _m = 25 μM) oxidase activity and was activated by addition of 0.1% (v/v) Triton-X-100 in the reaction mixture. Moreover, the laccase activity was increased 2.6-fold by the addition of 10 mM copper sulfate; the enzyme was totally inhibited by ethylenediaminetetraacetic acid (5 mM), suggesting that this laccase is a metal-dependant one. Decolorization activity of some synthetic dyes (methylene blue, methyl green, toluidine blue, Congo red, methyl orange, and pink) and the industrial effluent (SITEX Black) was achieved by the bacteria S. maltophilia AAP56 in the LB growth medium under shaking conditions.     

Investigating the New Interaction Model between Cu(II) and PVA by the Proton Relaxation Method

An aqueous PVA-Cu²⁺ solution at pH = 3 is in the hydrated form and becomes green at pH ? 6 with a decrease in viscosity. The structure of the copper ion is suggested to be that of a polynuclear complex at pH > 6. For the green solution the polynuclear chains of the copper complex are believed to be surrounded by the PVA chains with the hydrophobic backbones facing toward the inside and the hydrophilic OH groups oriented toward the outside facing the bulk water. The proton spin-lattice relaxation rate 1/T_1p and the spin-spin relaxation rate 1/T₂ of CH and CH₂ in PVA and H₂O for aqueous PVA-Cu²⁺ solutions at pH = 3, can be explained by the two site exchange model in the region of the fast exchange limit. The dipolar correlation time τ_c is dominated by the reorientational process with a dipolar correlation time of 2.11 × 10^?11 s. When the pH rises from pH=3 to pH=12.5, the variation of 1/T₁p and 1/T₂p of CH and CH₂ in PVA with Cu²⁺ ion concentration in aqueous PVA-Cu²⁺ solution at pH=12.5 can be explained in terms of the relaxation by an inclusive model of the polynuclear copper complex and PVA. Furthermore, the frequency (or field) dependence of 1/T₁p, 1/T₂p of CH in PVA for aqueous PVA-Cu²⁺ solution at pH = 12.5 suggests that the dipolar relaxation is dominated by the electron-spin relaxation with the electron spin relaxation time T_1e = 1 ? 2 × 10^?10 s. The invariance of 1/T₁p and 1/T₂p of H₂O with the variation of the Cu²⁺ ion concentration in aqueous PVA-Cu²⁺ solution at pH = 12.5 supports the hypothesis that the water is not directly bound to the Cu²⁺ ion.     

Overproduction of Laccase and Pectinase by Microbial Associations in Solid Substrate Fermentation

The growth and the enzymatic production of two microbial fungal associations were studied: Aspergillus niger and Fusarium moniliforme and Trametes versicolor and Aspergillus niger. The synergistic interrelations between the species of the first mixed culture increased the biosynthesis of α-amylase and pectinase. T. versicolor and A. niger proved to be compatible partners in the overproduction of the enzyme laccase, whose synthesis surpassed 8.4 times the enzymatic level in the monoculture, with both of the mixed microbial populations cocultivation facilitating the amplified synthesis of enzymes rather than their growth acceleration. A further proof of the presence of synergism established by the cultures was the enzyme volumetric productivities in both of the mixed microbial cultures, which increased parallel to the rise in the combined biomass synthesis. The competent selection of compatible partners can adjust the desired enzymatic levels and compositions in mixed fungal systems aimed at a number of specified designations. Thus, a very high level of laccase production (97,600 IU/g dry weight) was achieved. The chosen fungal strains produce a variety of different enzymes, but first microbial association produces mainly amylase and pectinase, necessary for their growth, and second association produces mainly laccase and pectinase.     

溶胶-凝胶法固定生物活性物质的研究进展

综述了近年来溶胶一凝胶(Sol-gel)技术在生物活性物质固定化方面的应用和进展。蛋白质、酶、抗体(抗原)、细胞及微生物均可被包埋于Sol-gel玻璃中。包埋后,这些生物活性物质仍保持其生物活性和光谱性质,有望成为实用的生物催化剂和生物传感器。     

载体型电化学传感器对有毒过渡金属离子Pb2+的检测

设计并制备了一种基于高分子聚合物(甲基丙烯酸甲酯-丙烯酸丁酯共聚物,MMA-BA)和碳纳米复合材料的固接离子选择性电极来检测水中铅离子Pb~(2+).电极使用高聚物MMA-BA作为传感膜的骨架,利用其优良的性质以及多壁碳纳米管的作用显著提高电极的传感性质.电极具有在低检测范围内的响应的特性,可将其应用在饮用水的实际检测中.     

Purification and Characterization of Laccase Secreted by <Emphasis Type="Italic">L. lividus</Emphasis>

The culture conditions for maximum secretion of laccase by Loweporus lividus MTCC-1178 have been optimized. The laccase from the culture filtrate of L. lividus MTCC-1178 has been purified to homogeneity. The molecular weight of the purified laccase is 64.8 kDa. The enzymatic characteristics like K _m, pH, and temperature optimum using 2,6-dimethoxyphenol have been determined and found to be 480 μM, 5.0, and 60 °C, respectively. The K _m values for other substrates like catechol, m-cresol, pyrogallol, and syringaldazine have also been determined and found to be 230, 210, 320, and 350 μM, respectively.     

Diffusion and Immobilization Mechanisms in Zeolites Studied by ZLC Chromatography

Zero Length Column chromatography was used to study mass transfer in zeolites involving coupled diffusion and immobilization mechanisms. A modeling based on Volterra integral equation technique was utilized to simulate sorption and desorption kinetic curves and compare results of the simulations with experimentally obtained curves. This approach was applied to analyze sorption kinetics in the model system: toluene/silicalite-1 (75°C–178°C). The system generally shows a non-Fickian behavior and can be described by diffusion coupled with immobilization.An erratum to this article can be found at     

Engineering of Laccase CueO for Improved Electron Transfer in Bioelectrocatalysis by Semi-Rational Design

Copper efflux oxidase (CueO) from Escherichia coli is a special bacterial laccase due to its fifth copper binding site. Herein, it is discovered that the fifth Cu occupancy plays a crucial and favorable role of electron relay in bioelectrocatalytic oxygen reduction. By substituting the residues at the four coordinated positions of the fifth Cu, 11 beneficial variants are identified with ≥2.5-fold increased currents at −250 mV (up to 6.13 mA cm⁻²). Detailed electrocatalytic characterization suggests the microenvironment of the fifth Cu binding site governs the electrocatalytic current of CueO. Additionally, further electron transfer analysis assisted by molecular dynamics (MD) simulation demonstrates that an increase in localized structural stability and a decrease of distance between the fifth Cu and the T1 Cu are two main factors contributing to the improved kinetics of CueO variants. It may guide a novel way to tailor laccases and perhaps other oxidoreductases for bioelectrocatalytic applications.