Optimization of Endoglucanase Production from Fomitopsis pinicola Mycelia

Fomitopsis pinicola 균사체로부터 Endoglucanase의 최적생산

The culture conditions to maximize the production of endoglucanase (EC 3.2.1.4) from the brown rot fungus Fomitopsis pinicola MKACC 54347 mycelia were investigated. Among the tested media for endoglucanase production, Mandel's mineral salts medium (MSM; 1% cellulose, 0.1% peptone, 0.14% $(NH_4)_2SO_4$, 0.03% urea, 0.2% $KH_2PO_4$, 0.03% $MgSO_4{\cdot}7H_2O$, 0.03% $CaCl_2$, and 0.1% trace metal solution (19.8 mM $FeSO_4$, 13.0 mM $MnSO_4$, 12.2 mM $ZnSO_4$, and 15.4 mM $CoCl_2$)) produced the highest activity of the enzyme. To optimize the medium composition for enzyme activity, the effects of various carbon, nitrogen, phosphorus, and inorganic sources were investigated in MSM. Maximal enzyme production was accomplished using a medium containing 2% carboxymethyl cellulose (CMC), 2% yeast extract, 0.2% $KH_2PO_4$, 0.03% $MnSO_4$, and 0.3% trace metal solution. Different physiological conditions, like incubation period and temperature, were also examined to assess their influence on enzyme production. Enzyme production from F. pinicola reached its highest level after cultivation for 8 days at $25^{\circ}C$. Nondenaturing polyacrylamide gel electrophoresis (PAGE), followed by the endoglucanase activity staining using CMC as the substrate, was performed to identify the endoglucanase under the culture conditions studied. Zymogram analysis of the culture supernatant revealed an endoglucanase band with a molecular mass of 52 kDa. The optimum pH and temperature for enzyme activity were $55^{\circ}C$ and pH 5.0, respectively.

소나무잔나비버섯(Fomitopsis pinicola MKACC 54347)의 균사체로부터 endoglucanase를 생산하기 위한 최적 배양조건을 조사하였다. 복합배지 중 MSM이 가장 높은 endoglucanase 활성을 나타내었으며, MSM의 성분과 농도를 각각 2.0% CMC, 2.0% yeast extract, 0.2% $KH_2PO_4$, 0.03% $MnSO_4$ 및 0.3% trace metal 용액으로 첨가하였을 때 효소활성이 가장 우수하였다. 따라서 F. pinicola로부터 endoglucanase를 생산하기 위한 최적 배지조건은 2.0% CMC, 2.0% yeast extract, 0.2% $KH_2PO_4$, 0.03% $MnSO_4$ 및 0.3% trace metal 용액이다. 이상의 배지를 사용하여 배양온도 $25^{\circ}C$에서 8일 동안 배양하였을 때 최대로 효소 활성이 나타내는 것을 확인하였다. CMC를 기질로 사용한 activity staining의 결과를 통해 F. pinicola 균사체의 endoglucanase 활성여부를 확인할 수 있었으며 그 분자량이 43-70 kDa 임을 확인하였고, 배양액 중의 효소활성의 최적 pH와 온도는 각각 pH 5.0과 $55^{\circ}C$인 것으로 나타났다.