• Journal of the Korean Wood Science and Technology
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• 제29권3호
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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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• 제33권1호
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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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• 제43권3호
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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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• 제24권5호
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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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• 제29권3호
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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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• 한국생물공학회 2001년도 추계학술발표대회
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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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• 제17권2호
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• pp.189-194
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• 2002

Trametes sp.에서 생산되는 laccase는 CNBr-activated Sepharose-4B(CAS4B)에 고정화 되었고, 염료의 연속적인 분해를 위하여 테스트되었다. Laccase는 CAS4B에 효율적으로 고정화되었고, CAS4B에 고정화 된 laccase는 pH, 열, 단백질 구조적인 안정성 면에서 상당히 증가하였다. CAS4B에 고정화 된 accase의 최적 pH는 5, 온도는 4$0^{\circ}C$로서 free laccase와 비교하여 변화가 없었다 기질로서 Reactive Blue 19를 사용하였을 때 free laccase와 고정화 laccase의 $K_{m}$ ($\mu$mol/mL) 값은 각각 0.34와 2.0이었고,V$_{max}$($\mu$mol/mL.min) 값은 각각 0.12, 0.1이었다. Repeated-batch 반응에서 효소의 안정성과 높은 분해 효율 만족하는 조건은 pH 5, 3$0^{\circ}C$이였다. 또한 HBT에 의한 효소의 불활성은 크게 나타나지 않았다. Packed-bed reactor에서 최적으로 운전되었을 때 100 $\mu$M Reactive Blue 19과 0.1 mM HBT가 존재하는 50 $\mu$M Acid Red 57의 연속적인 분해에서 30시간 후에도 분해 효율이 70%로 유지되었다. 고정화 laccase는 Packed-bed reactor에서 azo 염료의 연속적인 분해를 매우 안정적으로 수행하였다.다.

• 전기화학회지
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• 제2권1호
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• pp.31-37
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• 1999

구리이온을 함유하는 효소인 laccase(Rhus vernicifera)를 self-assembly technique을 이용하여 금전극 표면에 고정시킨 후 표면의 특성을 관찰하고 반응을 살펴보았다. laccase는 diphenol, diamine등을 산소에 의해 산화시킬 수 있는 oxidoreductase이다. 이 경우 산소는 peroxide나 superoxide 등의 중간체 생성없이 물까지 직접 4전자 환원이 일어난다. $\beta-mercaptopropionate$를 이용하여 금전극 표면에 음전하를 띤 self-assembled monolayer를 형성시킨 후, 중성용액에서 양 전하를 띤 laccase(pI=9)를 정전기적 인력에 의해 고정시킨 후, 순환 전압-전류법에 의한 실험으로 전극표면에 고정되었음을 확인하였다. 또한, 낮은 주사속도에서 흐른 전하량으로부터 surface coverage를 계산하여 전극표면에 효소가 monolayer로 덮여 있음을 확인하였다. laccase가 고정된 전극을 laccase의 기질인 ABTS(2,2-azino-bis-(3-ethylbenzthioline-6-sulfonic acid) 용액에 담그면 ABTS가 산화되는 것으로부터 고정된 laccase가 활성을 가지고 있음을 확인하였고, 그 효소효과는 $4^{\circ}C$에서 $2\~3$일 동안 지속됨을 관찰하였다. 앞서 구한 surface coverage로부터 고정된 효소의 양을 알 수 있어서, 표면에 고정된 laccase가용액상의 laccase에 비하여 $10\~15\%$정도만의 효소효과를 유지하고 있음을 알 수 있었다. 또한, laccase의 산소의 전기화학적 환원 촉매로서의 역할에 대하여 용액상에서와 전극표면에 고정시켰을 경우에 비교하여 보았는데, 두 경우 다 전자전달체가 없이는 산소환원의 촉매로 작용하지 않고, $Fe(CN)_6^{3-}$를 전자전달체로 사용한 경우에 산소환원의 촉매로 작용함을 알 수 있었다. 이러한 산소환원촉매로서의 역할이 laccase로부터 기인한다는 것은 억제제인 azide를 이용한 실험으로 다시 한 번 확인할 수 있었다.

• Journal of Electrochemical Science and Technology
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• 제8권2호
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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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• 제18권6호
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• pp.564-566
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• 1997