목재화학 및 실험

목재부후균 (Wood Rot Fungi)

1. 목재부후균 접종 및 배양

2. 목재부후균을 이용한 organosolv lignin 분해

조교 : 홍창영

■ Incubation

● White rot fungi (백색부후균)

○ Ceriporia lacerata (CEL) : 1조

○ Stereum hirsutum * (STH*) : 2조

○ Fomitopsis insularis (FOI): 3조

● Medium : Kirk's medium (SSC medium, nitrogen-limited)

● Incubation : 28℃, Organosolv lignin 접종 후, 14일 배양

■ Biodegradation of Organosolv lignin using UV-Vis spectrophotometer

● 0.5 M NaOH 25ml + 배양액 ▶ Homogenizing ▶ 원심분리 ▶ 상등액 ▶ 흡광도 측정

● 200-400 nm에서 흡광도의 변화 확인

■ Analysis of enzyme activity

● Laccase

○ 2,2'-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) was used as substrate for the determination of laccase activity in extracellular culture media.  The reaction mixture for laccase was prepared 0.85 mL of 0.2 M lactate buffer (pH 4.5) with 50 μL of ABTS (0.08 g/L) and 100 μL of culture medium to make total volume of 1 mL. The absorbance of that mixture at 420 nm wavelength was determined after 3 min of incubation at room temperature, and the enzyme activity was expressed using the equation below (ε420 = 36,000 M-1cm-1).

● Manganese-dependent Peroxidase (MnP)

○ MnP assay was performed by using ABTS as substrate for measuring enzyme activity. The reaction mixture for MnP was prepared 0.8 mL of 0.2 M lactate buffer (pH 4.5) with 50 μL of ABTS, 33 μL of MnSO4, 100 μL of culture medium, and 17 μL of H2O2. The absorbance at 420 nm wavelength was measured after 30 min. The enzyme activity was expressed using the equation below (ε420 = 36,000 M-1cm-1).

Enzyme activity (unit / mL) =	ΔAbsorbance × 106 × Total volume(L)
	ΔTime × ε420 × Sample volume(mL)

■ Report

● UV-Vis spectrophotometer의 원리, 사용 분야 등 전반적인 것을 조사할 것. (단, copy & paste로 판단되면 감점. 조사한 자료를 숙독한 후 자신이 이해할 수 있는 만큼 풀어서 서술할 것)

● 세 가지 fungi의 측정한 효소 역가를 조합하여 여러 가지 방향에서 비교추론해볼 것.

● Laccase, Manganese-dependent peroxidase (MnP)가 어떠한 기작으로 기질에 작용하는지 조사해올 것.

※ 반드시 논문 reference 추가할 것