• Journal of Microbiology
• /
• 제43권6호
• /
• pp.555-560
• /
• 2005

Laccase is one of the ligninolytic enzymes of white rot fungus Trametes versicolor 951022, a strain first isolated in Korea. This laccase was purified 209-fold from culture fluid with a yield of $6.2\%$ using ethanol precipitation, DEAE-Sepharose, Phenyl-Sepharose, and Sephadex G-100 chromatography. T. versicolor 951022 excretes a single monomeric laccase showing a high specific activity of 91,443 U/mg for 2,2'-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) as a substrate. The enzyme has a molecular mass of approximately 97 kDa as determined by SDS-PAGE, which is larger than those of other laccases reported. It exhibits high enzyme activity over broad pH and temperature ranges with optimum activity at pH 3.0 and a temperature of $50^{\circ}C$. The $K_m$ value of the enzyme for substrate ABTS is $12.8{\mu}M$ and its corresponding $V_{max}$ value is 8125.4 U/mg. The specific activity and substrate affinity of this laccase are higher than those of other white rot fungi, therefore, it may be potentially useful for industrial purposes.

• Journal of Microbiology and Biotechnology
• /
• 제24권12호
• /
• pp.1673-1678
• /
• 2014

The interactive inhibitory effects of pH and chloride on the catalysis of laccase from Trametes versicolor were investigated by studying the alteration of inhibition characteristics of sodium chloride at different pHs for the oxidation of 2,2'-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid). At pH 3.0, the addition of sodium chloride (50 mM) brought about a 40-fold increase in $K{_m}^{app}$ and a 4-fold decrease in $V_{max}{^{app}}$. As the pH increased to 7.0, the inhibitory effects of sodium chloride became significantly weakened. The mixed-inhibition mechanism was successfully used to quantitatively estimate the competitive and uncompetitive inhibition strengths by chloride at two different pHs (pH 3.0 and 6.0). At pH 3.0, the competitive inhibition constant, $K_i$, was 0.35 mM, whereas the uncompetitive inhibition constant, $K{_i}^{\prime}$, was 18.1 mM, indicating that the major cause of the laccase inhibition by chloride is due to the competitive inhibition step. At a higher pH of 6.0, where the inhibition of the laccase by hydroxide ions takes effect, the inhibition of the laccase by chloride diminished to a great extent, showing increased values of both the competitive inhibition constant ($K_i=23.7mM$) and uncompetitive inhibition constant ($K{_i}^{\prime}=324mM$). These kinetic results evidenced that the hydroxide anion and chloride share a common mechanism to inhibit the laccase activity.

• 미생물학회지
• /
• 제42권3호
• /
• pp.235-237
• /
• 2006

먹물버섯은 버섯 시원체로부터 버섯이 성숙되는 과정에서 자가소화가 일어나 먹물이라 불리는 검은 액체를 생성한다. 이 과정에서 멜라닌을 생성하는 laccase, 균류 세포벽 성분의 하나인 키틴을 분해하는 chitinase의 관련을 분석하고자 Northern hybridization 방법을 이용하여 유전자의 발현을 분석하였다. 시원체가 생성되고 버섯이 성숙되어 먹물을 생성하는 시기에 따라 멜라닌색소 생성 효소인 laccase와 킨틴분해효소인 chitinase의 발현이 증가하는 것이 확인되었다.

• 미생물학회지
• /
• 제52권3호
• /
• pp.249-253
• /
• 2016

먹물버섯의 하나인 Coprinellus congregatus는 생활사 동안 여러 종의 laccase 효소를 생성한다. 균사 끝 효소와 버섯시원체 효소 및 sclerotium (균핵) 효소들은 모두 이 균의 분화와 관련되었다. 이핵체 균사를 산성 액체배지(pH 4.0-4.5)에 접종하면 새로운 laccase가 합성되어 분비된다. 이 laccase 유전자의 프로모터의 어느 부분이 산 충격의 신호에 관련된 단백질이 결합하는가 분석하기 위하여 녹색형광단백질(green fluorescent protein, GFP) 유전자를 laccase 프로모터 2.0 kb 다음에 연결하고, 이를 형질전환 벡터인 pBARGEM7-1에 삽입함으로써 발현벡터를 구축하였다. 이 promoter-GFP 조합의 5'-region부터 차례로 제거한 짧은 길이의 이 발현벡터를 먹물버섯 교배형 a1균과 a2균에 형질전환 방법으로 도입시키고 phosphinothricin 저항성으로 형질전환체들을 선발하였다. 선발된 형질전환체 a1 (a1TF)과 a2 (a2TF)를 서로 교배하여 동형접합(homozygotic) 이핵체 형질전환체를 만들었다. 이들을 산성 액체배지에서 36시간 배양하고 균체를 모아 confocal microscope를 사용하여 형광을 분석하였다. Laccase 유전자의 전체 프로모터(2.0 kb)를 가진 발현벡터(F0-GFP)를 도입한 동형접합 형질전환체에서는 형광을 보였으나, 그 보다 짧은 길이(1.29 kb 이하)의 프로모터를 가진 형질전환체에서는 형광이 나타나지 않았다. 이 결과에 근거하여 먹물버섯의 산 충격에 대한 신호를 받는 부위가 laccase 유전자 프로모터의 -2.0 kb ~ -1.29 kb 사이에 있을 것으로 추정한다.

• 한국환경농학회지
• /
• 제25권3호
• /
• pp.211-216
• /
• 2006

Coriolus hirsutus LD-1 균주의 리그닌분해효소 활성과 몇몇 염료의 탈색능을 조사하였다. 백색부후균인 LD-1 균주는 laccase(16,388.9 U/L)와 manganese- dependent peroxidase (19.81 U/L)는 생산하였으나 lignin peroxidase를 생산하지 않았다. 균주를 염료와 함께 8일간 배양했을때 염료 RBBR과 염료 BB의 탈색율은 각각 70.2%와 98%로 나타났다. Manganese-dependent peroxidase는 8일간 배양 중 효소 활성은 매우 낮은 반면 laccase는 지속적으로 생산되어 대단히 높은 활성을 나타내었다. 백색부후균인 Coriolus hirsutus LD-1에 의한 염료의 탈색은 주로 laccase에 의한 것으로 사료되었다.

• Journal of the Korean Wood Science and Technology
• /
• 제17권3호
• /
• pp.52-60
• /
• 1989

This study was carried out to know the possibility of simultaneous utilization of laccase from white-rot fungus with cellulase on enzymatic hydrolysis of cellulosic substrate from autohydrolyzed oak wood. Laccases from 3 white-rot fungi, Pleurotus ostreatus. Ganoderma lucidum, and Phanerochaete chrysosporium, were isolated, purified and measured their activities. The highest activity was shown in Pleurotus ostreatus and the lowest in Phanerochaete chrysosporium. Laccase from Pleurotus ostreatus has optimum pH of 5.94, Km value of 3.209 mM and appeared to be stable at relatively wide pH range, 4.7-8.72. Temperature stability showed that 60% activity was preserved after 40 minutes at $50^{\circ}C$. Laccase from Ganoderma lucidum reached to the maximum activity during 15-20 day incubation. This enzyme has optimum pH of 6.45, Km value of 6.71 mM and pH range of 5.0-9.0 for stabilization. 95% activity was preserved at $30^{\circ}C$ and 58% activity at $50^{\circ}C$. Concerned to the enzymatic hydrolysis of cellulosic substrate with both enzymes, cellulase and laccase, simultaneously, mixed culture filtrates and mycellium extracts were shown higher hydrolysis rates than those of Trichoderma viride. There were no significant differences in the extent of hydrolysis among various mixed culture filtrates and mycellium extracts.

• 미생물학회지
• /
• 제30권2호
• /
• pp.78-82
• /
• 1992

담자성 효모인 Trimorphomyces papilionaceous 는 생장에 thiamine 을 요구하는 최적배양온도는 25.deg.C 이며 넓은 범위의 pH (4.0-7.0) 에서 잘 자랐고 최소배지네 여러가지 탄소원을 가하고 (1%) 배양하여 균의 생장과 laccase 의 역가를 조사하였다. Glucose, frutose, mannose, sucrose 와 xylose 배지에서 OD 0.8 이상으로 자랐고 (A group), galactose 와 gluconate 의 경우 OD 0.3-0.6 (B group), 그리고 arbinose, lactose 와 pyruvate 배지에서 CD0.1-0.2 를 보였다. (C-group), Ribose, acetate, citrate, lactate 와 oxaloctate 는 이용하지 못하였다. Laccase 는 C group 배지에서 가장 높은 역가를 보였고 (8 u 이상), B group 배지에서 4-6 unit, A group 배지의 경우 가장 낮은 1.5 unit 이하를 보였다. 이균을 glucose 와 arabinose 가 혼합된 배지에 배양할 경우 laccase 는 catabolite repression 에 의하여 조절되었다.

• Journal of Microbiology and Biotechnology
• /
• 제24권5호
• /
• pp.639-647
• /
• 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.

• 한국균학회지
• /
• 제15권2호
• /
• pp.99-170
• /
• 1987

Coriolus versicolor가 생산(生産)하는 laccase 생산(生産) 조건(條件)과 그 특성(特性)을 검토하여 다음과 같은 결과를 얻었다. Laccase 생산(生産)은 영양적 최적(最適) 조건(條件)은 indulin AT1%, peptone 0.3%, $KH_2PO_4$ 0.1%, $MgSO_4$ 0.02%, $CuSO_4$ 0.1mg%, thiamine HCI 0.005 mg%였다. Laccase 생산(生産)의 최적(最適) 온도(溫度)는 $25^{\circ}C$ 최적(最適) pH는 5.0이었고 배양기간(培養期間)은 20日이었다. Laccase 작용(作用) 최적(最適) pH는 4.6이었고 최적온도(最適最適)는 $40^{\circ}C$였으며 pH 안정성은 $40^{\circ}C$에서 15분간 보존(保存)하였을 때 pH4.6에서 가장 안정하였다. 열안정성(熱安定性)은 $40^{\circ}C$ 이하에서 는 비교적 안정하였으나 그 이상 올라갈수록 열에 민감한 반응(反應)을 보였고 sodium azide 1mM차 KCN 1mM에서 완전 저해(沮害)되었다.

• Journal of Microbiology
• /
• 제42권2호
• /
• pp.94-98
• /
• 2004

Phenanthrene is a three-ring polycyclic aromatic hydrocarbon and commonly found as a pollutant in various environments. Degradation of phenanthrene by white rot fungus Trametes versicolor 951022 and its laccase, isolated in Korea, was investigated. After 36 h of incubation, about 46％ and 65％ of 100 mg/l of phenanthrene added in shaken and static fungal cultures were removed, respectively. Phenanthrene degradation was maximal at pH 6 and the optimal temperature for phenanthrene removal was 30$^{\circ}C$. Although the removal percentage of phenanthrene was highest (76.7％) at 10 mg/1 of phenanthrene concentration, the transformation rate was maximal (0.82 mg/h) at 100 mg/L of phenanthrene concentration in the fungal culture. When the purified laccase of T. versicolor 951022 reacted with phenanthrene, phenanthrene was not transformed. The addition of redox mediator, 2,2'-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) or 1-hydroxybenzotriazole (HBT) to the reac-tion mixture increased oxidation of phenanthrene by laccase about 40％ and 30％, respectively.