Biocatalitic Oxidation of 2,3,6-Trimethylphenol Over Immobilized Horseradish Peroxidase in Nonaqueous Media

In this work various samples of horseradish peroxidase immobilized over organic and inorganic supports were synthesized and studied in 2,3,6-trimethylphenol biocatalitic oxidation. Silica, alumina and commercial samples of polymers MN-100, Sepabeads EC-HA were used as the carriers for horseradish peroxidase. Different methods of immobilization including treatment with chitosan, glutaric dialdehyde and carbodiimide were used to enhance catalytic activity. For synthesized catalysts optimal conditions for 2,3,6-trimethylphenol (main intermediates in vitamin E synthesis) oxidation providing high degree of conversion (more than 95%) and high selectivity to target products (more then 98%) were found. Physicochemical investigations (FTIR spectroscopy, XPS, nitrogen physosorption) of optimal biocatalytic systems were provided.     

Influence of varying surface hydrophobicity of chitosan membranes on the adsorption and activity of lipase

The hydrophobic surface modification of chitosan membranes was performed using the amidating reaction of amino groups on a membrane surface with stearic acid activated by 1‐ethyl‐3‐(3‐dimethylaminopropyl)‐carbodiimide hydrochloride (EDC) and N‐hydroxysuccinimide (NHS). The varying surface hydrophobicity of chitosan membranes was achieved by changing the degree of amidation and evaluated by the water contact angle analysis and the adsorption experiment of the hydrophobic dye, Rose Bengal. The effects of the surface hydrophobicity of chitosan membranes on the adsorption behaviors, activity and stability of Candida rugosa lipases were investigated. The experimental results suggested that the increased surface hydrophobicity of chitosan membranes improved the adsorption capacity and activity of the immobilized lipase. The modified chitosan membranes with 30.36% amidation exhibited the maximum activity retention of 83.87%. In addition, a desirable thermal stability was also achieved for the adsorbed lipase. © 2009 Wiley Periodicals, Inc. J Appl Polym Sci, 2010     

Synthesis of polyaniline using horseradish peroxidase immobilized on plasma‐functionalized polyethylene surfaces as initiator

The doping of semiconducting conjugated polymers (e.g., polyaniline, PANI) can result in the elimination of the bandgap, leading to high electrical conductivities (comparable to metals). Doped PANI is totally insoluble and thus nonprocessable, which considerably limits its practical applications. Synthesis of PANI using immobilized horseradish peroxidase (HRP) as a catalyst in aqueous solutions can open up additional possibilities for applications through the direct synthesis of controlled‐thickness PANI layers on various substrate surfaces. The RF plasma‐enhanced surface functionalization of polyethylene and the covalent immobilization of HRP are discussed, and the polymerization of aniline initiated by immobilized HRP is presented. The nature of plasma‐grafted surface functionalities on the substrate surfaces and the formation of PANI are demonstrated using X‐ray photoelectron spectroscopy and attenuated total reflectance Fourier transform IR spectroscopy. The molecular weight distribution of PANI is evaluated with gel permeation chromatography, and the activity of the immobilized enzyme is monitored using UV spectroscopy. © 2003 Wiley Periodicals, Inc. J Appl Polym Sci 88: 369–379, 2003     

以层柱黏土为载体固定辣根过氧化物酶

引 言 辣根过氧化物酶(HRP)能在较宽的温度、pH值、污染物浓度和盐度范围内将多种芳香性化合物催化氧化为酚氧自由基,它们之间可聚合生成溶解性较差的聚合物从溶液中沉淀出来,便于采用混凝法对其进行去除[1-2].许多学者将该酶用于含酚废水处理中,取得了较好的效果.然而,传统酶催化方法中使用的大多是溶液酶,不仅不能循环使用以节省费用,而且易受废水中其他污染物的影响,稳定性差,加上酶的费用较高,因此酶法在废水处理中尚未得到有效推广应用[3].国内外学者研究发现酶固定化后稳定性大大提高,可重复或连续使用,这样不仅降低了废水处理的成本,而且还避免了蛋白质的污染等问题[4].     

Removal of Persistent Sulfamethoxazole and Carbamazepine from Water by Horseradish Peroxidase Encapsulated into Poly(Vinyl Chloride) Electrospun Fibers

Enzymatic conversion of pharmaceutically active ingredients (API), using immobilized enzymes should be considered as a promising industrial tool due to improved reusability and stability of the biocatalysts at harsh process conditions. Therefore, in this study horseradish peroxidase was immobilized into sodium alginate capsules and then trapped into poly(vinyl chloride) electrospun fibers to provide additional enzyme stabilization and protection against the negative effect of harsh process conditions. Due to encapsulation immobilization, 100% of immobilization yield was achieved leading to loading of 25 μg of enzyme in 1 mg of the support. Immobilized in such a way, enzyme showed over 80% activity retention. Further, only slight changes in kinetic parameters of free (K_m = 1.54 mM) and immobilized horseradish peroxidase (K_m = 1.83 mM) were noticed, indicating retention of high catalytic properties and high substrate affinity by encapsulated biocatalyst. Encapsulated horseradish peroxidase was tested in biodegradation of two frequently occurring in wastewater API, sulfamethoxazole (antibiotic) and carbamazepine (anticonvulsant). Over 80% of both pharmaceutics was removed by immobilized enzyme after 24 h of the process from the solution at a concentration of 1 mg/L, under optimal conditions, which were found to be pH 7, temperature 25 °C and 2 mM of H₂O₂. However, even from 10 mg/L solutions, it was possible to remove over 40% of both pharmaceuticals. Finally, the reusability and storage stability study of immobilized horseradish peroxidase showed retention of over 60% of initial activity after 20 days of storage at 4 °C and after 10 repeated catalytic cycles, indicating great practical application potential. By contrast, the free enzyme showed less than 20% of its initial activity after 20 days of storage and exhibited no recycling potential.     

Application of chitosan‐entrapped β‐galactosidase in a packed‐bed reactor system

The model enzyme β‐galactosidase was entrapped in chitosan gel beads and tested for hydrolytic activity and its potential for application in a packed‐bed reactor. The chitosan beads had an enzyme entrapment efficiency of 59% and retained 56% of the enzyme activity of the free enzyme. The Michaelis constant (K_m) was 0.0086 and 0.011 μmol/mL for the free and immobilized enzymes, respectively. The maximum velocity of the reaction (V_max) was 285.7 and 55.25 μmol mL^?1 min^?1 for the free and immobilized enzymes, respectively. In pH stability tests, the immobilized enzyme exhibited a greater range of pH stability and shifted to include a more acidic pH optimum, compared to that of the free enzyme. A 2.54 × 16.51‐cm tubular reactor was constructed to hold 300 mL of chitosan‐immobilized enzyme. A full‐factorial test design was implemented to test the effect of substrate flow (20 and 100 mL/min), concentration (0.0015 and 0.003M), and repeated use of the test bed on efficiency of the system. Parameters were analyzed using repeated‐measures analysis of variance. Flow (p < 0.05) and concentration (p < 0.05) significantly affected substrate conversion, as did the interaction progressing from Run 1 to Run 2 on a bed (p < 0.05). Reactor stability tests indicated that the packed‐bed reactor continued to convert substrate for more than 12 h with a minimal reduction in conversion efficiency. © 2003 Wiley Periodicals, Inc. J Appl Polym Sci 91: 1294–1299, 2004     

Effect of water‐miscible solvents on the organic solvent‐resistant tyrosinase from Streptomyces sp. REN‐21

The effect of various water‐miscible organic solvents (ethanol, methanol, acetone, acetonitrile, N,N‐dimethylformamide (DMF) and dimethylsulfoxide (DMSO)) on the kinetics of 4‐tert‐butylcatechol (tBC) oxidation in the presence of different samples of organic solvent‐resistant tyrosinase (OSRT) has been studied. In contrast to mushroom tyrosinase the enzyme shows a high relative stability in solutions of organic solvents and increased activity toward the bulky and hydrophobic substrate, tBC, in respect to catechol. Rates of the studied OSRT‐catalyzed reactions are however reduced by the presence of organic solvents and for all studied samples of OSRT decrease exponentially with the content of an organic solvent. The effect has been satisfactorily described by the effect of organic solvents on the thermodynamic activity of tBC. The correlation of the inhibition parameters with the hydrophobicity of a particular solvent (log P), its intrinsic molar volume, V_i, and the Dimroth–Reichardt parameter, E_T(30), are shown. The results allow also the prediction of OSRT activity in aqueous solutions of water‐miscible organic solvents. Copyright © 2003 Society of Chemical Industry     

超声波对固定化辣根过氧化物酶活性影响研究

为了研究超声波对固定化酶活性的影响,以固定化辣根过氧化物酶为对象,研究了不同超声波处理条件(超声功率,超声时间)以及超声条件下催化体系的pH、温度对固定化酶活性的作用。同时对超声波处理后固定化酶活的重复利用性进行测定。结果表明,超声波处理对提高固定化酶在高温、强酸碱条件下催化活性有一定帮助,最佳处理条件为:超声波功率50 W,超声时间30 min,pH 8,温度35℃,在此条件下,与未经超声波处理相比,固定化酶活性提高了17.6%,固定化酶重复利用性增强,经7次使用后,固定化酶催化活性是未经处理的1.8倍。     

Biomimetic Multienzyme Complexes Based on Nanoscale Platforms

Inspired by the widely present multienzyme complexes in nature that enable highly cooperative catalytic mechanisms, we designed a biomimetic dual‐functionalized nanoparticle‐based platform for colocalizing multiple enzymes. The use of nanoscale materials together with a novel sequential colocalization approach with two model enzymes [glucose oxidase (GOX) and horseradish peroxidase] resulted in a 100% increase in the overall conversion rate compared to the equivalent amount of free enzymes in solution and a physical mixture of individual immobilized enzymes on nanoparticles. GOX is an important enzyme used in glucose biosensors for diagnostics. Colocalizing GOX with peroxidase allows for colorimetric visualization of the peroxide formed that enables monitoring glucose levels in solution. This platform can be readily applicable to other multienzyme systems as well. © 2012 American Institute of Chemical Engineers AIChE J, 59: 355–360, 2013     

Preparation of chitosan‐coated magnetite nanoparticles and application for immobilization of laccase

In this study, immobilization of laccase (L) enzyme on magnetite (Fe₃O₄) nanoparticles was achieved, so that the immobilized enzyme could be used repeatedly. For this purpose, Fe₃O₄ nanoparticles were coated and functionalized with chitosan (CS) and laccase from Trametes versicolor was immobilized onto chitosan‐coated magnetic nanoparticles (Fe₃O₄‐CS) by adsorption or covalent binding after activating the hydroxyl groups of chitosan with carbodiimide (EDAC) or cyanuric chloride (CC). For chitosan‐coated magnetic nanoparticles, the thickness of CS layer was estimated as 1.0–4.8 nm by TEM, isoelectric point was detected as 6.86 by zeta (ζ)‐potential measurements, and the saturation magnetization was determined as 25.2 emu g^?1 by VSM, indicating that these nanoparticles were almost superparamagnetic. For free laccase and immobilized laccase systems, the optimum pH, temperature, and kinetic parameters were investigated; and the change of the activity against repeated use of the immobilized systems were examined. The results indicated that all immobilized systems retained more than 71% of their initial activity at the end of 30 batch uses. © 2011 Wiley Periodicals, Inc. J Appl Polym Sci, 2011     

Removal of alkylphenols by the combined use of tyrosinase immobilized on ion‐exchange resins and chitosan beads

Mushroom tyrosinase was covalently immobilized on a poly(acrylic acid)‐type, weakly acidic cation‐exchange resin (Daiaion WK10, Mitsubishi Chemical Corp., Tokyo, Japan) with 1‐ethyl‐3‐(3‐dimethylaminopropyl)carbodiimide hydrochloride salt as a water‐soluble carbodiimide. Ion‐exchange resins immobilized with tyrosinase were packed in one column, and crosslinked chitosan beads prepared with epichlorohydrin were packed in another column. The enzymatic activity was modified by covalent immobilization, and the immobilized tyrosinase had a high activity in the temperature range of 30–45°C and in the pH range of 7–10. When solutions of various alkylphenols were circulated through the two columns packed with tyrosinase‐immobilized ion‐exchange resins and crosslinked chitosan beads at 45°C and pH 7 (the optimum conditions determined for p‐cresol), alkylphenols were effectively removed through quinone oxidation with immobilized tyrosinase and subsequent quinone adsorption on chitosan beads. The use of chemically crosslinked chitosan beads in place of commercially available chitosan beads was effective in removing alkylphenols from aqueous solutions in shorter treatment times. The removal efficiency increased with an increase in the amount of crosslinked chitosan beads packed in the column because the rate of quinone adsorption became higher than the rate of enzymatic quinone generation. The activity of tyrosinase was iteratively used by covalent immobilization on ion‐exchange resins. One of the most important findings obtained in this study is the fact that linear and branched alkylphenols suspected of weak endocrine‐disrupting effects were effectively removed from aqueous solutions. © 2009 Wiley Periodicals, Inc. J Appl Polym Sci, 2010     

Immobilized fungal biocatalysts for the production of cellulase complex hydrolyzing renewable plant feedstock

We discuss characteristics of the directional formation of samples of heterogeneous biocatalysts based on immobilized cells of different microscopic fungi that are characterized by high productivity of cellulases with different substrate specificities (endoglucanases, exoglucanases, and beta-glucosidases). Samples of immobilized cells characterized by maximum productivity of the enzymes of cellulase complex are selected based on our study of the catalytic and operating characteristics of the designed biocatalysts. It is found that a biocatalyst based on Aspergillus terreus spores immobilized in polyvinyl alcohol cryogel is the best of the ones available. It is shown for the first time that the developed biocatalyst retains a high level of productivity for the full complex of cellulases when using various substrate inducers of enzyme biosynthesis, such as birch and oak sawdust, and rice and wheat straw. We demonstrate the possibility of efficiently using cellulase complexes obtained as a result of the functioning of immobilized cells in the saccharification of various cellulose-containing agricultural wastes and the conversion of the obtained sugars to organic solvents (ethanol, butanol) considered to be promising alternative fuels. The concentrations of organic solvents in media with immobilized cells are considerably higher than those found for free cells of the same microorganisms.     

Enhanced activities of lipase pretreated with organic solvents

The catalytic activities of lipases derived from Pseudomonas sp and pretreated with various organic solvents were investigated. The activity of the solvent‐pretreated lipase was greater than that of native lipase in both the esterification reaction in an organic medium and the hydrolysis reaction in an aqueous medium. With esterification calalysed by pretreated lipase, the product, benzyl octanoate, was detected without time‐lag. Conversions at equilibrium state were correlated with the hydrophobicities of the solvents used. In the hydrolysis reaction, most pretreated lipases yielded increased acid production compared with native lipase. A linear correlation was observed between the solvent hydrophobicity and the relative initial reaction rate of the hydrolysis reaction when using pretreated lipases. © 2001 Society of Chemical Industry     

固定化辣根过氧化物酶的电化学和电催化特性

用海藻酸钠(SA)将辣根过氧化物酶(HRP)固定到热裂解石墨电极表面,制备了HRP-SA膜修饰电极。研究发现包埋在SA膜中的辣根过氧化物酶可与电极直接传递电子,在缓冲溶液中可得到一对辣根过氧化物酶辅基血红素Fe(Ⅲ)/Fe(Ⅱ)电对的可逆氧化还原峰。其式电势随溶液pH值增加而负移,且呈线性关系,这说明辣根过氧化物酶的电子传递过程伴随有质子的转移。并考察了HRP-SA膜修饰电极对氧气、双氧水、一氧化氮的电催化性质。     

Synthesis of NAD polystyrene microspheres and application as a carrier of glucoamyrase immobilization

Polystyrene microspheres with a small amount of acrylic acid (200 : 1) incorporated were synthesized by nonaqueous phase dispersion (NAD) polymerization to investigate the feasibility of use as a carrier for immobilized enzyme. Seeded emulsion polymerization was then carried out to obtain larger cross-linked polystyrene spheres, the structure of which was expected to yield a favorable environment to keep the original activity of the immobilized enzyme. Glucoamylase, a particular enzyme catalyzing the hydrolysis of oligosaccharides to glucose, was immobilized through peptide bonding using a carbodiimide activating agent. Thirty percent of the enzyme activity at maximum was retained relative to free glucoamylase. The immobilized enzyme remained active after 4 months, although its poor resistance to heat was not improved. © 1994 John Wiley & Sons, Inc.     

非水相细胞催化研究进展

非水相细胞催化可提高水不溶化合物溶解度,改变热力学平衡以利于产物合成,是生物催化的重要组成部分。本文介绍了非水相细胞催化体系中溶剂和催化剂的筛选,如lgP值法和以产物高萃取率为目标的计算机辅助分子设计,以及催化剂的溶剂耐受性;综述了提高非水相细胞催化活性的方法,包括极端微生物的筛选和构建、细胞固定化等,以及非水相细胞催化反应在香料、药物及药物中间体等方面的应用现状;最后从生物学和工程学角度展望了非水相细胞催化研究的努力方向。     

Stability of a chitosan layer deposited onto a polyethylene surface

A two‐step procedure was applied to obtain antimicrobial films; this procedure involved a corona treatment of the polyethylene (PE) surface and its chemical activation with 1‐ethyl‐3‐[3‐dimethylaminopropyl] carbodiimide hydrochloride and N‐hydroxysuccinimide, and this led to the covalent bonding of chitosan on the PE surface. Electrochemical methods were used to investigate the stability of the deposited chitosan layer. The potentiometric and polyelectrolyte titrations showed that some amount of chitosan desorbed faster from the surface until equilibrium was reached and also that the grafted chitosan layer was more stable than the physically adsorbed one. The chitosan immobilized on the PE surface exhibited the expected antibacterial activity when tested against three bacteria, which included two Gram‐negative bacteria, Salmonella enteritidis and Escherichia coli, and one Gram‐positive bacterium, Listeria monocytogenes. © 2013 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 130: 2444–2457, 2013     

Immobilization strategy of accessible transmission for trypsin to catalyze synthesis of dipeptide in mesoporous support

Immobilized trypsin in mesoporous silica foams was used to catalyze dipeptide synthesis in hydrophilic organic solvent instead of soluble form. The area surface of nano support was measured. The catalytic activity, coupled yield and kinetic characterization of immobilized trypsin were examined. Bz-Arg-OEt was chosen as the acyl donor with Lys-OH as the nucleophile. The trypsin-catalyzed synthesis condition was optimized, such as catalytic temperature, pH, reaction time, physical properties and content of organic solvents, together with the added enzyme amount. The immobilized trypsin showed 112.8% of residual activity with 91.9% of coupled yield, and the kinetic parameters exhibited accessibility for transmission. The product yield of 5.8% was reached at the optimum conditions for enzymatic synthesis of dipeptide: 800 mg of wet immobilized trypsin (200 mg/g support) was used in Tris-HCl buffer (0.1 mol/L, pH 8.0) containing 80% (v/v) ethanol solvents for 6 h of reaction time at 35 °C. This attempt of immobilized strategy for trypsin in nanopores renders the possibility of wide application of inorganic nano-sized support in catalytic synthesis process, which can avoid usage of large amounts of organic solvents in washing steps by chemical methods and reduce the tedious purification process of its soluble form.     

Immobilization of β‐Glucosidase BglC on Decanedioic Acid‐Modified Magnetic Nanoparticles

β‐Glucosidase BglC from Thermobifida fusca was efficiently immobilized on decanedioic acid‐modified magnetic nanoparticles (DMNP). Interestingly, the immobilized BglC showed much higher organic solvent stability than the free enzyme, indicating that this enzyme immobilized on DMNP should be especially suitable for catalyzing reactions in organic solvents. Additionally, the immobilized BglC also showed better thermal stability, storage stability, and mechanical stability than the free counterpart. Moreover, the reuse times were satisfactory and even crude plant materials (e.g., rice husk and corn cob) were used as substrates, which should be valuable for the decomposition of natural organic materials and the full utilization of bioresources.     

Enzyme and chemical encapsulation in polymeric microcapsules

Polypyrrole microcapsules (prepared via the template method) were used for immobilization of both enzymatic and chemical catalytic systems. Enzymes immobilized include glucose oxidase, catalase, trypsin, subtilisin, and alcohol dehydrogenase. The chemical catalytic system investigated consisted of immobilized Pd nanoparticles for the catalysis of hydrogen peroxide decomposition. Microcapsules loaded with glucose oxidase (GOD) were found to have higher enzymatic activity than that of GOD-loaded thin films, a competing encapsulation method. Trypsin was used to explore the possible leakage of small proteins from the capsules; no leakage was observed. Subtilisin was used to show that these microcapsules can be used in nonaqueous solvents. The effect of the capsule wall thickness on the rate of the enzymatic reaction was also explored. © 1996 John Wiley & Sons, Inc.