• Journal of the Korean Wood Science and Technology
• /
• v.24 no.4
• /
• pp.74-81
• /
• 1996

리그닌분해능(分解能)이 높은 균주(菌株)로 선발(選拔)된 Coriolus versicolor-13 (CV-13), LKY-7 및 LKY-12세 균주(菌株)에 대하여 균체외(菌體外) laccase 생산(生産)을 검토(檢討)하였다. Glucose-peptone broth에서 균체외(菌體外) laccase활성(活性)은 CV-13의 경우 3일 이상배양후(以上培養後)에 나타났고 LKY-7과 LKY-12균주(菌株)의 laccase 활성(活性)은 배양(培養) 2일째에 검출(檢出)되었다. 탄소원(炭素源)으로서는 maltose가 glucose와 비슷한 laccase 생산효과(生産效果)를 나타냈고 질소원(窒素源)으로서는 유기태질소(有機態窒素)가 무기태(無機態) 질소(窒素)보다 효과적(效果的)이었다. Laccase 유도물질(誘導物質)로서는 2,5-Xylidine이 가장 우수하였으며 1mM 이하(以下)의 농도(濃度)에서는 유도효과(誘導效果)가 크게 나타났으나 1.5mM 이상(以上)의 농도(濃度)에서는 laccase생산(生産)이 억제(抑制)되었고, 균사생장(菌絲生長) 초기(初期)에 첨가(添加)하는 것이 효과적(效果的)으로 나타났다. SDS-PAGE 후, CV-13 균주(菌株)의 균체외(菌體外) 단백질(蛋白質)에서는 약 69, 66, 25, 23, 19kDa 크기의 laccase band가 5개 나타났고 LKY-7 균주(菌株)에서는 27kDa과 19kDa 크기의 2개 band가, LKY-12 균주(菌株)에서는 22, 20, 17kDa 크기의 laccase band가 3개 나타났다.

• Microbiology and Biotechnology Letters
• /
• v.34 no.3
• /
• pp.228-234
• /
• 2006

The culture conditions were investigated to maximize the production of laccase from Fomitella fraxinea mycelia. Among the tested media, mushroom complete medium (MCM) showed the highest production of the enzyme. The optimum culture medium was 2% dextrose, 0.4% $(NH_4)_{2}HPO_4$, 0.05% $Na_{2}HPO_{4}{\cdot}7H_{2}O$, and 0.05% KCl as carbon, nitrogen, phosphorus, and inorganic salt sources respectively. SDS-PAGE followed by laccase activity staining using 2,6-djmethoxyphenol as the substrate was performed to identify the laccase activity under culture conditions studied. Zymogram analysis of the culture supernatant showed a laccase band with a molecular mass of 50 kDa. The enzyme production from F. fraxinea was reached to the highest level after the cultivation for 10 days at $25^{\circ}C$ and initial pH 8. The enzyme activity of the culture supernatant was most active at $50^{\circ}C$ and pH 5.

• Journal of Life Science
• /
• v.25 no.6
• /
• pp.673-679
• /
• 2015

The culture conditions needed to maximize the production of laccase from Pholiota highlandensis mycelia were investigated. Among the tested media for laccase production, Coriolus versicolor medium (CVM; 2% dextrose, 0.4% peptone, 0.6% yeast extract, 0.046% KH₂PO₄, 0.1% K₂HPO₄, 0.05% MgSO₄·7H₂O) showed the highest activity for the enzyme. Then, to optimize culture conditions for laccase activity, the influences of various carbon, nitrogen, phosphorus, and inorganic salt sources in CVM were investigated. The optimum culture medium was 2% fructose, 0.4% peptone with 0.6% yeast extract, 0.05% NaH₂PO₄, and 0.05% MgSO₄·7H₂O as carbon, nitrogen, phosphorus, and inorganic salt sources, respectively. Several aromatic compounds in the medium enhanced laccase activity to varying degrees. Guaiacol induced maximum laccase production, yielding 114.1 U/ml laccase activity after cultivation for 11 days at 25℃. The optimum pH and temperature for laccase production were 8.0 and 35℃, respectively. Native polyacrylamide-gel electrophoresis (PAGE) followed by laccase-activity staining with 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) as the substrate was performed to identify the presence of laccase under the optimum conditions studied. Zymogram analysis of the supernatant culture showed an enzymatic band with a molecular mass of about 90 kDa.

• Bulletin of the Korean Chemical Society
• /
• v.23 no.7
• /
• pp.985-989
• /
• 2002

For efficient biopulping process, very active and stable lignase is essential. Laccase is one of the best enzyme in terms of environmentally benign processes, since the enzyme uses oxygen as an oxidant to degrade lignin and produces no hamful prod ucts. We could purify a laccase homogeneously from Cerrena unicolor in a very active state. It shows characteristic absorption feature with blue band at λmax = 604 ㎚. Molecular weight of the enzyme is 57,608 which could be accurately determined by MALDI/TOF MS. The enzyme has 2.8 copper ions per enzyme implying apoenzymes might exist together. The enzyme is active in lignin degradation and the activity increases 4 times in the presence of ABTS as a mediator.

• Microbiology and Biotechnology Letters
• /
• v.37 no.1
• /
• pp.62-68
• /
• 2009

The culture conditions to maximize the production of laccase (EC 1.10.3.2) from Fomitopsis pinicola mycelia were investigated. Among the tested media for the enzyme production, mushroom complete medium (MCM ; 2% dextrose, 0.2% peptone, 0.2% yeast extract, 0.05% $KH_2PO_4$, and 0.05% $MgSO_4{\cdot}7H_2O$) showed the highest activity of the enzyme. To optimize the culture condition for the laccase activity, influence of various carbon and nitrogen sources was investigated in MCM. Among various carbon and nitrogen sources, 2% glucose and 0.4% peptone showed the highest production of the enzyme, respectively. For the phosphorus and inorganic source, 0.05% $NaH_2PO_4$ and 0.05% $CaCl_2$ were best for the enzyme activity. The enzyme production was reached to highest level after the cultivation for 8 days at $25^{\circ}C$. Native polyacrylamide gel electrophoresis (PAGE) followed by the laccase activity staining using 2, 2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) as the substrate was performed to identify the laccase under culture conditions studied. Zymogram analysis of the culture supernatant showed a laccase band with molecular mass of 52 kDa. The optimum pH and temperature for the enzyme activity were $80^{\circ}C$ and pH 3.0.

• Korean Journal Plant Pathology
• /
• v.13 no.4
• /
• pp.225-231
• /
• 1997

Activities of polygalacturonase, laccase, and intra- and extra-cellular $\beta$-glucosidase produced by 20 Botrytis cinerea isolates in liquid culture media containing cucumber cell was as a carbon source were measured and their relationships to the pathogenicity were analyzed. No significant correlations between these enzyme activities and the pathogenicity of B. cinerea were found. Mycelial growth rate on Bayendamm media, however, was higthly correlated with the pathogenicity (r=0.522) anong these isolates. Immuno-blot analysis of the culture filtrate using antibody against against exo-polygalacturonase revealed that only one band with molecular weight of 66 kDa was detected amone 34 tested isolates. It appears that these enzymes may not be primary factors in dermining the pathogenicity of B. cinerea.

• Journal of the Korean Wood Science and Technology
• /
• v.21 no.3
• /
• pp.87-93
• /
• 1993

This study was undertaken to investigate the characteristics and the productivities of lignin olytic enzymes: laccase (Lac) and Mn-dependent peroxidase (MnP) from Coriolus versicolor and Lentinus edodes respectively. Enzymes were isolated from cultural filterates and purified according to the standard methods. These enzymes showed one band in SDS-PAGE and their molecular weights were found 62,000 and 45,000 dalton respectively. Polyclonal antibodies against Lac and MnP were raised against mouse. In the ELISA (enzyme-linked immunosorbent assay), Lac and MnP-antiserum produced a strong positive reaction with Lac and MnP antigen($A_{405}$=2.50 and 3.53 respectively). The sera to negative (S/N) ratio was determined by the dividing the mean absorbance of antibodies by the corresponding diluted samples from normal mouse serum. The sera produced showed 2 times more positive reaction in S/N ratio than negative sera.

• Journal of Microbiology and Biotechnology
• /
• v.24 no.4
• /
• pp.545-555
• /
• 2014

It has been previously demonstrated that laccases exhibit great potential for use in several industrial and environmental applications. In this paper, two laccase isoenzyme genes, lccB and lccC, were cloned and expressed in Pichia pastoris GS115. The sequence analysis indicated that the lccB and lccC genes consisted of 1,563 and 1,584 bp, and their open reading frames encoded 520 and 527 amino acids, respectively. They had 72.7% degree of identity in nucleotides and 86.7% in amino acids. The expression levels of LccB and LccC were up to 32,479 and 34,231 U/l, respectively. The recombinant laccases were purified by ultrafiltration and $(NH_4)_2SO_4$ precipitation, showing a single band on SDS-PAGE, which had a molecular mass of 58 kDa. The optimal pH and temperature for LccB were 2.0 and $55^{\circ}C$ with 2,2'-azinobis-[ 3-ethylbenzthiazolinesulfonic acid (ABTS) as a substrate, whereas LccC exhibited optimal pH and temperature at 3.0 and $60^{\circ}C$. The apparent kinetic parameters of LccB were 0.43 mM for ABTS with a $V_{max}$ value of 51.28 U/mg, and the Km and $V_{max}$ values for LccC were 0.29 mM and 62.89 U/mg. The recombinant laccases were able to decolorize five types of dyes. Acid Violet 43 (100 g/ml) was completely decolorized by LccB or LccC (2 U/ml), and the decolorization of Reactive Blue KN-R (100 g/ml) was 91.6% by LccC (2 U/ml). Thus, the study characterizes useful laccase isoenzymes from T. versicolor that have the capability of being incorporated into the treatment of similar azo and anthraquinone dyes from dyeing industries.