Trametes velutina JS18 유래 멜라닌 탈색 효소의 생산, 정제 및 특성ㅍ
Production, Purification and Characterization of a Melanin Bleaching Enzyme from Trametes velutina JS18
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삼림지역의 고목에서 분리한 JS18 균주는 합성 멜라닌을탈색하는 세포 외 분비효소를 생산했다. JS18 균주의 internal transcribed spacer (ITS) 염기서열을 분석하고 계통학적으로 확인한 결과 본 균주는 Trametes velutina로 동정되었다. JS18 균주는 laccase 활성을 나타냈지만 manganese peroxidase 및 lignin peroxidase 활성은 나타내지 않았다. 본 균주를 회분배양한 결과 멜라닌 탈색 활성은 laccase 활성으로부터 유래하는 것으로 확인되었다. Laccase 유도인자로써 Syringic acid 및 CuSO4를 첨가하고 25℃에서 7일간배양한 결과 배양상등액에서 98 U/ml의 laccase 활성을 나타내었다. GYP 배지에서 배양한 T. velutina의 배양상등액에서 ammonium sulfate 침전, Hi-trap Q Sepharose 컬럼및 gel filtration을 이용하여 효소를 정제하였고, SDS-PAGE 에서 약 67 kDa의 분자량을 나타내었다. 정제된 효소의 멜라닌 탈색율은 효소 단독으로는 24 시간 만에 단지 4% 만을나타내는 반면, HBT의 존재 하에서는 80%로 향상되었다. 또한 1.5 mM HBT의 농도에서는 최대 81%의 멜라닌 탈색율을 나타내었다. 본 효소의 멜라닌 탈색에 대한 최적 pH 및온도는 각각 5.0와 37℃를 나타내었다. 본 연구에서는 T. velutina JS18 유래 laccase가 촉매하는 멜라닌 탈색 반응에서 redox mediator로써 HBT의 적용 가능성을 확인하였다.
Abstract
The JS18 strain was isolated from an old tree forest and produced extracellular enzymes that decolorize synthetic melanin. Phylogenetic analysis, based on the internal transcribed spacer (ITS) sequence, indicate that JS18 belongs to the Trametes velutina species. JS18 demonstrated laccase activity but no manganese peroxidase or lignin peroxidase activity. Batch culture indicated that the melanin decolorization activity of JS18 strain originated from the laccase. Syringic acid and CuSO4 induced maximum laccase production, yielding 98 U/ml laccase activity after cultivation for 7 days at 25℃. T. velutina secretes an extracellular laccase in GYP medium, and this enzyme was purified using (NH4)2SO4 precipitation, Hi-trap Q Sepharose columns and gel filtration. The molecular weight of the purified enzyme was estimated to be 67 kDa using sodium dodecyl sulfate polyacrylamide gel electrophoresis. This enzyme produced 80% of its melanin decolorization activity within the first 24 h of evaluation in the presence of 1-hydroxybenzotriazole (HBT), while only about 4% of the melanin was decolorized in the absence of the mediator. The greatest decolorization was observed at 1.5 mM/l HBT, which decolorized 81% of the melanin within the first 24 h. The optimum pH and temperature for this decolorization were found to be 5.0 and 37℃, respectively. Our results suggest the possibility of applying HBT induced T. velutina JS18 laccase-catalyzed melanin decolorization.
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