Decolorization of Kraft effluent by free and immobilized lignin peroxidases and horseradish peroxidase

Color removal from Kraft effluent by lignin peroxidase and horseradish peroxidase was compared. Free lignin peroxidase and horseradish peroxidase removed color from kraft effluent. Immobilization of lignin peroxidase type III, lyophilized fungal culture and horseradish peroxidase on CNBr-Sepharose 4B improved the decolorization by factor of 2.9, 4.5 and 2.6, respectively in 48 h. Lignin peroxidase type I was effective only in the immobilized form in decolorization. In general, the immobilized form all the studied systems exhibited an average value around of 30% polymer consumption and very little of depolymerization. Lignin peroxidases and lyophilized fungal culture were shown to have considerable potential for treating Kraft effluents.     

The treatment of chlorophenols with laccase immobilized on sol–gel-derived silica

Laccase, a so-called “blue-copper” oxidase, is able to oxidize a variety of organic compounds. Sol–gel derived silica glasses are frequently adopted as an immobilization method to improve the stability of enzymes and make them reusable. In this study, immobilization conditions were optimized to achieve improved embedding results. The thermal stability, reaction stability and storage stability were improved with the immobilized enzyme when compared to the free enzyme. 2,4-Dichlorophenol (DCP) and 2,4,6-trichlorophenol (TCP) were chosen as model compounds. The treatment of chlorophenols (CPs) by immobilized laccase demonstrated excellent removal and response stability. The affinity of TCP for immobilized laccase was higher than that of DCP. This finding leads to different removal efficiencies under variable conditions (reaction time, initial concentration, dosage of immobilized laccase and removal rate in mixed solution). By fitting the experimental data with the diffusion model of the degradation process, the degradation of CPs by immobilized laccase matches an intraparticle diffusion-controlled model.     

Immobilization of manganese peroxidase from Lentinula edodes and its biocatalytic generation of MnIII-chelate as a chemical oxidant of chlorophenols

Manganese peroxidase (MnP) purified from commercial cultures of Lentinula edodes was covalently immobilized through its carboxyl groups using an azlactone-functional copolymer derivatized with ethylenediamine and 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (EEDQ) as a coupling reagent. The tethered enzyme was employed in a two-stage immobilized MnP bioreactor for catalytic generation of chelated MnIII and subsequent oxidation of chlorophenols. Manganese peroxidase immobilized in the enzyme reactor (reactor 1) produced MnIII-chelate, which was pumped into another chemical reaction vessel (reactor 2) containing the organopollutant. Reactor 1-generated MnIII-chelates oxidized 2,4-dichlorophenol and 2,4, 6-trichlorophenol in reactor 2, demonstrating a two-stage enzyme and chemical system. H2O2 and oxalate chelator concentrations were varied to optimize the immobilized MnP's oxidation of MnII to MnIII. Oxidation of 1.0 mM MnII to MnIII was initially measured at 78% efficiency under optimized conditions. After 24 h of continuous operation under optimized reaction conditions, the reactor still oxidized 1.0 mM MnII to MnIII with approximately 69% efficiency, corresponding to 88% of the initial MnP activity.     

Controlled layer-by-layer immobilization of horseradish peroxidase.

Horseradish peroxidase (HRP) was biotinylated with biotinamidocaproate N-hydroxysuccinimide ester (BcapNHS) in a controlled manner to obtain biotinylated horseradish peroxidase (Bcap-HRP) with two biotin moieties per enzyme molecule. Avidin-mediated immobilization of HRP was achieved by first coupling avidin on carboxy-derivatized polystyrene beads using a carbodiimide, followed by the attachment of the disubstituted biotinylated horseradish peroxidase from one of the two biotin moieties through the avidin-biotin interaction (controlled immobilization). Another layer of avidin can be attached to the second biotin on Bcap-HRP, which can serve as a protein linker with additional Bcap-HRP, leading to a layer-by-layer protein assembly of the enzyme. Horseradish peroxidase was also immobilized directly on carboxy-derivatized polystyrene beads by carbodiimide chemistry (conventional method). The reaction kinetics of the native horseradish peroxidase, immobilized horseradish peroxidase (conventional method), controlled immobilized biotinylated horseradish peroxidase on avidin-coated beads, and biotinylated horseradish peroxidase crosslinked to avidin-coated polystyrene beads were all compared. It was observed that in solution the biotinylated horseradish peroxidase retained 81% of the unconjugated enzyme's activity. Also, in solution, horseradish peroxidase and Bcap-HRP were inhibited by high concentrations of the substrate hydrogen peroxide. The controlled immobilized horseradish peroxidase could tolerate much higher concentrations of hydrogen peroxide and, thus, it demonstrates reduced substrate inhibition. Because of this, the activity of controlled immobilized horseradish peroxidase was higher than the activity of Bcap-HRP in solution. It is shown that a layer-by-layer assembly of the immobilized enzyme yields HRP of higher activity per unit surface area of the immobilization support compared to conventionally immobilized enzyme.     

Catalytic activity of poly(acryloyl morpholine)-immobilized horseradish peroxidase in organic/aqueous solvent mixtures

The immobilization of horseradish peroxidase by covalent coupling within an expanded poly(acryloyl morpholine) gel network is described. The activity of the immobilized horseradish peroxidase was compared with that of the native enzyme in aqueous buffer and in buffered mixtures of dimethyl-formamide/water, ethanediol/water, methanol/water and tetrahydrofuran/water of varying solvent ratios at pH 6.1. On increasing the organic solvent concentration in the substrate solution, active immobilized enzyme retained its activity much better than an equivalent amount of the native enzyme. The oxidation of ferrocene (water-insoluble) and ferrocene derivatives to the corresponding ferricinium ions, was accomplished efficiently by the immobilized enzyme in buffered 50% methanol/water solution. The immobilized enzyme exhibited superior resistance to thermal denaturation.     

Effect of soil moisture on bioremediation of chlorophenol-contaminated soil

A chlorophenol-contaminated soil was tested for the biodegradability in a semi-pilot scale microcosm using indigenous microorganisms. More than 90% of 4-chlorophenol and 2,4,6-trichlorophenol, initially at 30 mg kg^–1, were removed within 60 days and 30 mg pentachlorophenol kg^–1 was completely degraded within 140 days. The chlorophenols were degraded more effectively under aerobic condition than under anaerobic condition. Soil moisture had a significant effect with the slowest degradation rate of chlorophenols at 25% in the range of 10–40% moisture content. At 25–40%, the rate of chlorophenol degradation was directly related to the soil moisture content, whereas at 10–25%, it was inversely related. Limited oxygen availability through soil agglomeration at 25% moisture content might decrease the degradation rate of chlorophenols.     

Degradation of Chlorophenols by Alcaligenes eutrophus JMP134(pJP4) in Bleached Kraft Mill Effluent

The ability of Alcaligenes eutrophus JMP134(pJP4) to degrade 2,4-dichlorophenoxyacetic acid, 2,4,6-trichlorophenol, and other chlorophenols in a bleached kraft mill effluent was studied. The efficiency of degradation and the survival of strain JMP134 and indigenous microorganisms in short-term batch or long-term semicontinuous incubations performed in microcosms were assessed. After 6 days of incubation, 2,4-dichlorophenoxyacetate (400 ppm) or 2,4,6-trichlorophenol (40 to 100 ppm) were extensively degraded (70 to 100%). In short-term batch incubations, indigenous microorganisms were unable to degrade such of compounds. Degradation of 2,4,6-trichlorophenol by strain JMP134 was significantly lower at 200 to 400 ppm of compound. This strain was also able to degrade 2,4-dichlorophenoxyacetate, 2,4,6-trichlorophenol, 4-chlorophenol, and 2,4,5-trichlorophenol when bleached Kraft mill effluent was amended with mixtures of these compounds. On the other hand, the chlorophenol concentration and the indigenous microorganisms inhibited the growth and survival of the strain in short-term incubations. In long-term (>1-month) incubations, strain JMP134 was unable to maintain a large, stable population, although extensive 2,4,6-trichlorophenol degradation was still observed. The latter is probably due to acclimation of the indigenous microorganisms to degrade 2,4,6-trichlorophenol. Acclimation was observed only in long-term, semicontinuous microcosms.     

Immobilization of tyrosinase on chitosan-clay composite beads

Tyrosinase was immobilized on glutaraldehyde crosslinked chitosan-clay composite beads and used for phenol removal. Immobilization yield, loading efficiency and activity of tyrosinase immobilized beads were found as 67%, 25% and 1400 U/g beads respectively. Optimum pH of the free and immobilized enzyme was found as pH 7.0. Optimum temperature of the free and immobilized enzyme was determined as 25-30 °C and 25 °C respectively. The kinetic parameters of free and immobilized tyrosinase were calculated using l-catechol as a substrate and K(m) value for free and immobilized tyrosinase were found as 0.93 mM and 1.7 mM respectively. After seven times of repeated tests, each over 150 min, the efficiency of phenol removal using same immobilized tyrosinase beads were decreased to 43%.     

BSA和PEG影响固定化辣根过氧化物酶HRP在有机相中活力

ＢＳＡ和ＰＥＧ可以有效地提高固定化辣根过氧化物酶（ＨＲＰ）在有机相中的活力。固定化酶活力的提高与试剂加入的顺序有密切的联系;不同载体对酶的影响不同,Ｇｅｌｉｔｅ,ａｌｕｍｉｎａ,ＸＡＤ－７,Ｋｉｓｅｌｇｅｌ和Ｆｌｏｒｉｓｉｌ为载体,分别以吸附法制备固定化酶。实验表明固定化过程中保护剂和酶的加入顺序与国家化酶活力密切相关,而这些载体的固定化效果又以Ｃｅｌｉｔｅ最佳,Ｆｌｏｒｉｓｉｌ最差。Ｆｌｏｒｉｓ     

Immunoaffinity layering of enzymes

A general procedure for the high yield immobilization of enzymes with the help of specific anti-enzyme antibodies is described. Polyclonal antibodies were raised against Aspergillus niger glucose oxidase and horseradish peroxidase in rabbits and the gamma globulin (IgG) fraction from the immune sera isolated by ammonium sulphate fractionation followed by ion-exchange chromatography. Immobilization of glucose oxidase and horseradish peroxidase was achieved by initially binding the enzymes to a Sepharose matrix coupled with IgG isolated from anti-(glucose oxidase) and anti-(horseradish peroxidase) sera, respectively. This was followed by alternate incubation with the IgG and the enzyme to assemble layers of enzyme and antibody on the support. The immunoaffinity-layered preparations obtained thus were highly active and, after six binding cycles, the amount of enzyme immobilized could be raised about 25 times over that bound initially. It was also possible to assemble layers of glucose oxidase using unfractionated antiserum in place of the IgG. The bioaffinity-layered preparations of glucose oxidase and horseradish peroxidase exhibited good enzyme activities and improved resistance to heat-induced inactivation. The sensitivity of a flow injection analysis system for measuring glucose and hydrogen peroxide could be remarkably improved using immunoaffinity-layered glucose oxidase and horseradish peroxidase. For the detection of glucose, a Clark-type oxygen electrode, constructed as a small flow-through cell integrated with a cartridge bearing immunoaffinity-layered glucose oxidase was employed. The hydrogen peroxide concentration was analysed spectrophotometrically using a flow-through cell and the layered horseradish peroxidase packed into a cartridge. The immunoaffinity-layered enzymes could be conveniently solubilized at acid pH and fresh enzyme loaded onto the support. Immunoaffinity-layered glucose oxidase was successfully used for the on-line monitoring of the glucose concentration during the cultivation of Streptomyces cerevisiae. Received: 16 November 1998 / Received revision: 22 March 1999 / Accepted: 26 March 1999     

Enhanced stabilization of mungbean thiol protease immobilized on glutaraldehyde-activated chitosan beads

A thiol protease purified from mungbean seedlings was immobilized on chitosan beads cross-linked with glutaraldehyde. The yield of the immobilized enzyme was maximum (～99%) at 1% concentration each of chitosan and glutaraldehyde. The immobilized enzyme showed reusability for 15 batch reactions. Immobilization shifted the optimum pH of the enzyme to a more acidic range and enhanced its stability both at acidic as well as alkaline pH values compared to the free enzyme. The stability of the enzyme to temperature and in aqueous non-conventional medium (ethanol and DMSO) was significantly improved by the immobilization process. The immobilized enzyme exhibited mass transfer limitation reflected by a higher apparent K_m value. This study produced an immobilized biocatalyst having improved characteristics and better operational stability than the soluble enzyme. The increase in stability in the presence of high concentrations of ethanol and DMSO may make it useful for catalyzing organic reactions such as trans-esterification and trans-amidation similar to other cysteine proteinases.     

Laccase stabilization by covalent binding immobilization on activated polyvinyl alcohol carrier

AIMS: Attempts were made to immobilize laccase from Panus conchatus. METHODS AND RESULTS: The laccase was immobilized on carboxylated polyvinyl alcohol (PVA) activated by N-hydroxysuccinimide (N-HSI) in aqueous solution at different pHs, temperatures, and with different reaction times. An optimum condition for laccase immobilization is at pH 3.2, 40 degrees C and 12 h, respectively. Immobilization of laccase increased optimal pH for reaction with 2, 2'-azinobis (3-ethylbenzthiazoline-6-solfonate) (ABTS) and pH stability. Immobilized laccase proved to be reacted consecutively 17 times with only a 50% decrease on activity and be used in removal of 2,4,6-trichlorophenol (TCP). CONCLUSIONS: It was possible to immobilize the laccase on carboxylated polyvinyl alcohol by activation with N-hydroxysuccinimide in HAc-NaAc buffer. The immobilized laccase is both stable and reusable. SIGNIFICANCE IMPACT OF THE STUDY: The results indicate that this immobilized laccase can be used in the removal of poisonous effluent from pulp bleaching mills.     

Peroxidase oxidation of phenols

Partially purified preparations of horseradish peroxidase were able to catalyze the effective transformation of such phenol compounds as phenol, o-chlorophenol, 2,4,6-trichlorophenol, pentachlorophenol (giving rise to the formation of polymer products insoluble in water), resorcinol, and thymol (giving rise to the formation of low-molecular-weight products). The following conditions were found to be optimal for peroxidase oxidation and provide the maximum extent of elimination of phenol compounds: temperature, 15-25 and 25-30 degrees C for phenol and chlorophenol compounds, respectively; molar ratio H2O2/phenol, 1:1; and transformation time, 1-3 h. Although effective transformation was observed within a broad range of pH, the efficiency of the process slightly increased at a pH from 6.0 to 7.5. It was suggested to carry out multiple peroxidase oxidations of phenols using partially purified peroxidase enclosed in a dialysis membrane bag placed into a solution of a phenol compound containing hydrogen peroxide.     

Immobilized white-rot fungal biodegradation of phenol and chlorinated phenol in trickling packed-bed reactors by employing sequencing batch operation

The biodegradation of phenol and 2,4,6-trichlorophenol (2,4,6-TCP) by immobilized white-rot fungal cultures was studied in pinewood chip and foam glass bead-packed trickling reactors. The reactors were operated in sequencing batch format. Removal efficiency increased over time and elevated influent phenol and 2,4,6-TCP (800 and 85 mg l(-1)) concentrations were removed by greater than 98% in 24-30 h batch cycles. Comparable performance between the packing materials was shown. Increased lignin peroxidase (LiP) activity was detected with the introduction of the compounds and optimum activity corresponded to optimum removal periods. Higher LiP activity (16.7-19 Ul(-1)) was detected in glass bead-packed reactor compared to wood chip reactor (0.2-5 Ul(-1)). The presence of Mn(2+) in the wood material possibly effected elevated manganese peroxidase (MnP) activity (0.3-5.8 Ul(-1)) compared to low to negligible activity in the glass bead reactor. Reactor performances are discussed in relation to sequencing batch operation and nutrient requirements necessary to induce and sustain fungal enzyme activity in inert vs. organic material packed systems.     

Effect of reaction conditions on phenol removal by polymerization and precipitation using Coprinus cinereus peroxidase

The quantitative relationships between removal efficiency of phenol and reaction conditions were investigated using Coprinus cinereus peroxidase. The most effective ratio of hydrogen peroxide to phenol was nearly 1/1 (mol/mol) at an adequate enzyme dose. 12.2 U of the enzyme was needed to remove 1 mg of phenol when our peroxidase preparation was used. At an insufficient peroxidase dose, the optimum pH value was 9.0, and lowering the reaction temperature led to the improvement of removal efficiency. At an excess peroxidase dose, almost 100% removal of phenol was obtained over a wide range of pH (5-9) and temperature (0-60 degrees C). Despite the presence of culture medium components, it was shown that Coprinus cinereus peroxidase had the same phenol polymerization performance as horseradish peroxidase or Arthromyces ramosus peroxidase.     

New immobilization method for enzyme stabilization involving a mesoporous material and an organic/inorganic hybrid gel

Horseradish peroxidase (HRP) was immobilized in a mesoporous material (folded sheets mesoporous materials, FSM-16) and then entrapped in organic/inorganic hybrid gel comprising various molar ratios of dimethyldimethoxysilane (DMDMOS)/tetramethoxysilane (TMOS). When pore size of FSM-16 materials is much larger than the diameters of horseradish peroxidase (HRP), the residual enzymatic activity after thermal treatment (70°C, 60 min) increased from 73 to 99%, and the oxidative conversion yield of 1,2-diaminobenzene in an organic solvent increased from 59 to 79% after 4 h and the level of leakage of immobilized HRP decreased from 6 to 1.5% on washing by secondary hybrid gel entrapment comprising a molar ratio of DMDMOS/TMOS=1:3. When pore size of FSM-16 materials just matches the diameter of the enzyme, the conversion yield in an organic solvent and the level of leakage of immobilized HRP did not change so much.     

改性壳聚糖固定化脂肪酶的研究

以化学改性后的壳聚糖为载体固定假丝酵母99-125脂肪酶,研究了不同的活化剂对壳聚糖表面羟基基团的活化程度,及以活化后壳聚糖为载体采用不同固定化方法对假丝酵母脂肪酶固定效果的影响。结果表明1-乙基-3-(3-甲基氨基)丙基碳二亚胺可有效的活化壳聚糖表面羟基,活化后的壳聚糖表面氨基与戊二醛偶联后形成的壳聚糖为良好的脂肪酶固定化载体,其固定脂肪酶的水解活力可高达86.8U/g。此外,还对影响固定化进程中的各种因素进行了研究,确定最优条件,比较了固定化前后酶的热稳定性、有机溶剂稳定性及最适反应温度。并考察了该固定化脂肪酶催化合成棕榈酸十六酯的操作稳定性,结果表明,连续反应16批之后棕榈酸十六酯的转化率仍能达到85%以上。     

Increased thermostability and phenol removal efficiency by chemical modified horseradish peroxidase

Horseradish peroxidase was modified by phthalic anhydride and glucosamine hydrochloride. The thermostabilities and removal efficiencies of phenolics by native and modified HRP were assayed. The chemical modification of horseradish peroxidase increased their thermostability (about 10- and 9-fold, respectively) and in turn also increased the removal efficiency of phenolics. The quantitative relationships between removal efficiency of phenol and reaction conditions were also investigated using modified enzyme. The optimum pH for phenol removal is 9.0 for both native and modified forms of the enzyme. Both modified enzyme could suffer from higher temperature than native enzyme in phenol removal reaction. The optimum molar ratio of hydrogen peroxide to phenol was 2.0. The phthalic anhydride modified enzyme required lower dose of enzyme than native horseradish peroxidase to obtain the same removal efficiency. Both modified horseradish peroxidase show greater affinity and specificity of phenol.     

Immobilization of horseradish peroxidase on β-cyclodextrin-capped silver nanoparticles: Its future aspects in biosensor application

This study aimed to work out a simple and high-yield procedure for the immobilization of horseradish peroxidase on silver nanoparticle. Ultraviolet–visible (UV-vis) and Fourier-transform infrared spectroscopy and transmission electron microscopy were used to characterize silver nanoparticles. Horseradish peroxidase was immobilized on β-cyclodextrin-capped silver nanoparticles via glutaraldehyde cross-linking. Single-cell gel electrophoresis (Comet assay) was also performed to confirm the genotoxicity of silver nanoparticles. To decrease toxicity, silver nanoparticles were capped with β-cyclodextrin. A comparative stability study of soluble and immobilized enzyme preparations was investigated against pH, temperature, and chaotropic agent, urea. The results showed that the cross-linked peroxidase was significantly more stable as compared to the soluble counterpart. The immobilized enzyme exhibited stable enzyme activities after repeated uses.     

Microbial removal of chlorinated phenols during aerobic treatment of effluents from radiata pine kraft pulps bleached with chlorine-based chemicals,with or without hemicellulases

  The removal of chlorophenolic compounds from kraft mill effluents bleached with chlorine (cBKME) or chlorine plus hemicellulases (bBKME) was studied in reactors of aerobic treatment lagoons. In these laboratory models, a stable microbial population removed biochemical oxygen demand at similar rates of the mill lagoon. Complete removal of nine chlorophenols and chloroguaiacols during microbial treatment of these effluents was detected by gas chromatography. Abiotic removal was only observed with 2,4-dichlorophenol and 2,4,5-trichlorophenol. There were no significant differences in degradative ability between microorganisms acclimated to grow in reactors fed with cBKME or bBKME. The latter had a lower content of adsorbable organic halogen and chlorophenols than cBKME. Microorganisms acclimated to cBKME or bBKME were only able to grow on phenol or guaiacol as sole carbon source. However, these microorganisms removed (0.1–0.5 mM) 4-chlorophenol, 2,4-dichlorophenol and 2,4-dichlorophenoxyacetate with BKME as primary carbon source. Under these conditions, 2,4,6- and 2,4,5-trichlorophenol, 4,5-dichloroguaiacol, 4,5,6-trichloroguaiacol and tetrachloroguaiacol were not removed. These results suggest that the microbial removal of bleaching chlorophenols and chloroguaiacols during aerobic treatment, probably takes place only because of their very low concentration (1–200 ppb) in BKME. Received: 12 February 1996 / Received revision: 10 June 1996 / Accepted: 22 June 1996