• Journal of the Korean Wood Science and Technology
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• v.29 no.3
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• pp.104-111
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• 2001

There are some evidence that active enzymatic proteins, e.g. fungal laccase, exist in the naturally occured soil humus. This study was performed to investigate the covalent binding of fungal laccase to the humic acid-iron complex, and to measure laccase activity of immobilized ones. Seven methods were adopted to form the covalent binding of fungal laccase with soil humic acids complexed with iron. Using these seven methods it was possible to change the dimension of spacer arm between laccase and support, and also to regulate the mode of covalent binding of this enzyme. The spacer arm was regulated from 2C to 11C. There was not observed any straight relationship between the spacer arm longitude and the laccase activity after immobilization, but the binding mode more effective than the former. Three out of the seven methods gave the high activity of immobilized laccase, and which active products of laccase immobilization was stable up to 10 days after the process. It is indicated that natural soil condition might be prevented the laccase activation by the toxic influence of some phenolic humic compounds. It was shown, for the first time, the possibilities to obtain the high activity of fungal laccase by binding to humic acids, and especially in complex with iron.

• Journal of Microbiology and Biotechnology
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• v.33 no.1
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• pp.127-134
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• 2023

Laccase activity is influenced by copper (Cu) as an inducer. In this study, laccase was immobilized on Cu and Cu-magnetic (Cu/Fe₂O₄) nanoparticles (NPs) to improve enzyme stability and potential applications. The Cu/Fe₂O₄ NPs functionally activated by 3-aminopropyltriethoxysilane and glutaraldehyde exhibited an immobilization yield and relative activity (RA) of 93.1 and 140%, respectively. Under optimized conditions, Cu/Fe₂O₄ NPs showed high loading of laccase up to 285 mg/g of support and maximum RA of 140% at a pH 5.0 after 24 h of incubation (4℃). Immobilized laccase, as Cu/Fe₂O₄-laccase, had a higher optimum pH (4.0) and temperature (45℃) than those of a free enzyme. The pH and temperature profiles were significantly improved through immobilization. Cu/Fe₂O₄-laccase exhibited 25-fold higher thermal stability at 65℃ and retained residual activity of 91.8% after 10 cycles of reuse. The degradation of bisphenols was 3.9-fold higher with Cu/Fe₂O₄-laccase than that with the free enzyme. To the best of our knowledge, Rhus vernicifera laccase immobilization on Cu or Cu/Fe₂O₄ NPs has not yet been reported. This investigation revealed that laccase immobilization on Cu/Fe₂O₄ NPs is desirable for efficient enzyme loading and high relative activity, with remarkable bisphenol A degradation potential.

• Journal of Microbiology
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• v.43 no.3
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• pp.301-307
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• 2005

The feasibility of laccase production by immobilization of Pleurotus ostreatus 1804 on polyurethane foam (PUF) cubes with respect to media composition was studied in both batch and reactor systems. Enhanced laccase yield was evidenced due to immobilization. A relatively high maximum laccase activity of 312.6 U was observed with immobilized mycelia in shake flasks compared to the maximum laccase activity of free mycelia (272.2 U). It is evident from this study that the culture conditions studied, i.e. biomass level, pH, substrate concentration, yeast extract concentration, $Cu^{2+}$ concentration, and alcohol nature, showed significant influence on the laccase yield. Gel electrophoretic analysis showed the molecular weight of the laccase produced by immobilized P. ostreatus to be 66 kDa. The laccase yield was significantly higher and more rapid in the packed bed reactor than in the shake flask experiments. A maximum laccase yield of 392.9 U was observed within 144 h of the fermentation period with complete glucose depletion.

• Journal of Microbiology and Biotechnology
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• v.24 no.5
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• pp.639-647
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• 2014

Laccases have a broad range of industrial applications. In this study, we immobilized laccase on $SiO_2$ nanoparticles to overcome problems associated with stability and reusability of the free enzyme. Among different reagents used to functionally activate the nanoparticles, glutaraldehyde was found to be the most effective for immobilization. Optimization of the immobilization pH, temperature, enzyme loading, and incubation period led to a maximum immobilization yield of 75.8% and an immobilization efficiency of 92.9%. The optimum pH and temperature for immobilized laccase were 3.5 and $45^{\circ}C$, respectively, which differed from the values of pH 3.0 and $40^{\circ}C$ obtained for the free enzyme. Immobilized laccase retained high residual activities over a broad range of pH and temperature. The kinetic parameter $V_{max}$ was slightly reduced from 1,890 to 1,630 ${\mu}mol/min/mg$ protein, and $K_m$ was increased from 29.3 to 45.6. The thermal stability of immobilized laccase was significantly higher than that of the free enzyme, with a half-life 11- and 18-fold higher at temperatures of $50^{\circ}C$ and $60^{\circ}C$, respectively. In addition, residual activity was 82.6% after 10 cycles of use. Thus, laccase immobilized on $SiO_2$ nanoparticles functionally activated with glutaraldehyde has broad pH and temperature ranges, thermostability, and high reusability compared with the free enzyme. It constitutes a notably efficient system for biotechnological applications.

• Journal of the Korean Wood Science and Technology
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• v.29 no.3
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• pp.112-122
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• 2001

Laccase enzymes from Cerrena unicolor and Trametes versicolor were immobilized on the activated glass beads (CPG), silica gel (SG) and soil (SL). The heterogeneous matrices were activated by ${\gamma}$-aminopropyltriethoxysilane (APTES) and glutaraldehyde (GA), and their surfaces were coated by keratin (KER) on activated or non-activated CPG, SG and SL. The laccase activities were tested in the aqueous solution for the native and immobilized preparations using different pH and temperature conditions. By keratin coating on supports, in the cases of CPG-KER and SL-KER, the immobilization yield was increased from about 80% to 90%. Moreover, much less protein was immobilized in keratin coated matrices than in inorganic ones alone (e.g. on CPG-KER 57.6%, whereas on CPG alone 80.6%). Laccase immobilization on keratin coated inorganic matrices was generally more effective than that of non-coated matrices. Concerned to pH dependency, the optima pH for immobilized laccases generally shifted towards to higher values, 5.5-5.8 and even 5.9 in the case of keratin for C. unicolor and from 5.3 to 5.7 for T. versicolor, respectively, and decreased less gradually both in acidic and alkaline regions. The immobilized laccase was more stable against thermal denaturation. This seems particularly true at $75^{\circ}C$ in the case of C. unicolor, where the activity of immobilized enzyme is > 50% higher than that of the free enzyme. For T. versicolor the respective values were $65^{\circ}C$, and 50%.

• 한국생물공학회:학술대회논문집
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• 2001.11a
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• pp.635-639
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• 2001

A laccase produced the Trametes sp. was immobilized on CNBr-activated Sepharose 4B(CS4B) and tested for repeated-batch and continuous decolorization of dye. After immobilization, the enzyme was active in wider pH and temperature range, and its heat stability was greatly improved compared to those of the free laccase. Immobilized laccase was efficient for both repeated-batch and contionuous decolorization.

• KSBB Journal
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• v.17 no.2
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• pp.189-194
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• 2002

Laccase produced from Trametes sp. was immobilized on CNBr-activated Sepharose-4B (CAS4B) and tested for degradation of azo dyes. Laccase was efficiently immobilized on CAS4B. Immobilization of laccase on CAS4B increased pH, thermal and proteolytic stabilities. Optimum pH and temperature of immobilized laccase were pH 3 and 40$\^{C}$, respectively as same as those of free laccase. The K$\_$m/($\mu$mol/ml) values of free and immobilized laccase for Reactive Blue 19 as the substrate were 0.34 and 2.07, respectively V$\_$max/($\mu$mol/mL$.$min) values of them were 0.12 and 0.1, respectively. In repeated batch reactions, conditions retained high stability and degradation of dye for immobilized laccase were pH 5 and 30$\^{C}$. HBT didn\\`t decrease highly activity of immobilized laccase. Immobilized laccase was very stable for degrading dyes continuously in a packed-bed reactor containing laccase immobilized on CAS4B. For continuous degradation of 100 $\mu$M Reactive Blue 19 and 50 $\mu$M Acid Red 57 in the presence of 0.1 mM HBT under optimum conditions, immobilized laccase retained 70% of degradation ability even after 30 hours.

• Journal of the Korean Electrochemical Society
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• v.2 no.1
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• pp.31-37
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• 1999

Copper-containig enzyme, laccase (Rhus vernicifera) was immobilized onto gold electrode using self-assembly technique and its surface properties and catalytic activities were examined. Laccase is an oxidoreductase capable to oxidize diphenols or diamines by 4-electron reduction of molecular oxygen without superoxide or peroxide intermediates. The electrode surface were modified by $\beta-mercaptopropionate$ to have a net negative charge in neutral solution and positively charged laccase (pI=9) was immobilized by electrostatic interaction. The successful immobilization was confirmed by cyclic voltammograms which showed typical surface-confined shapes and behaviors. The amount of charge to reduce the surface was similar to the charge calculated assuming the surface being covered by monolayer. The activity of the immobilized enzyme was tested by the capbility of oxidizing a substrate, ABTS (2,2-azine-bis-(3-ethylbenzthioline-6-sulfonic acid) and it was maintained for $2\~3$ days at $4^{\circ}C$. The immobilzed laccase showed about $10\~15\%$ activity compared to that in solution. The laccase-modified electrode showed the activity of elefoocatalytic reduction of oxygen in the presence of mediator, $Fe(CN)_6^{3-}$ The addtion of azide which is an inhibitor of laccase compeletly eliminated the catalytic current.

• Journal of Electrochemical Science and Technology
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• v.8 no.2
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• pp.87-95
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• 2017

Enzymatic fuel cells are promising low cost, compact and flexible energy resources. The basis of enzymatic fuel cells is transfer of electron from enzyme to the electrode surface and vice versa. Electron transfer is done either by direct or mediated electron transfer (DET/MET), each one having its own advantages and disadvantages. In this study, the DET and MET of laccase-based biocathodes are compared with each other. The DET of laccase enzyme has been studied using two methods; assemble of needle-like carbon nanotubes (CNTs) on the electrode, and CNTs/Nafion polymer. MET of laccase enzyme also is done by use of ceramic electrode containing, ABTS (2,2'-azino-bis [3-ethylbenzthiazoline-6-sulphonic acid]) /sol-gel. Cyclic voltammetric results of DET showed a pair of well-defined redox peaks at $200{\mu}A$ and $170{\mu}A$ in a solution containing 5and $10{\mu}M$ o-dianisidine as a substrate for needle-like assembled CNTs and CNTs-Nafion composite respectively. In MET method using sol-gel/ABTS, the maximum redox peak was $14{\mu}A$ in the presence of 15 M solution o-dianisidine as substrate. The cyclic voltammetric results showed that laccase immobilization on needle-like assembled CNTs or CNTs-Nafion is more efficient than the sol-gel/ABTS electrode. Therefore, the expressed methods can be used to fabricate biocathode of biofuel cells or laccase based biosensors.

• Bulletin of the Korean Chemical Society
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• v.18 no.6
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• pp.564-566
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• 1997