섬유소분해효소 생산증진을 위한 Penicillium verruculosum의 균주개량

Strain Improvement of Penicillium verruculosum for High Cellulase Production by Induced Mutation

천연섬유소 분해활성이 우수하고 그 효소 유도기구도 Trichoderma reesei와는 다른 Penicillium verruculosum F-3을 모균주로 사용하여 돌연변이 처리에 의한 유전적 개량을 시도함으로써 cellulase 생산성이 증진된 조절변이주를 얻고자 변이주 유도조건 변이주에 의한 cellulase 생성조건을 검토하였다. 한천평판상에서의 변이주의 선택분리 효율을 향상시키기 위하여 각종 colony 소형화제의 영향을 검토한 바 Oxgall을 배지에 1.5% 첨가하였을 때가 가장 좋았다. Cellulase 고생산성 변이주 선정의 한 지표로서 대사산물 억제의 해제를 선택했다. P. verruculosum F-3은 glucose 또는 glycerol 농도 1% 이상에서 본 효소생성이 억제되었다. 변이주 유도조건으로서 UV조사의 경우는 19분 처리로 약 0.2%, NTG 처리의 경우는 200$\mu\textrm{g}$/$m\ell$ 농도로 1시간처리로 48%의 생존율을 나타냈다. 변이처리 한 균주를 여지붕괴도, cellulose agar plate에서의 clear zone의 크기, cellulose powder 배지로 배양한 조효소액의 여지분해활 성을 조사하여 우수균주로서 UV-9, UV-10 및 NTG-3을 최종 선발했다. 각종 탄소원을 함유하는 배지에서의 cellulase 생산성을 조사한 바 KC-M-W 배지로 배양한 UV-9, UV-10 및 NTG-3의 여지분해 활성은 친주보다 각각 34%, 55%, 41% 증가되었으나, UV-9 및 NTG-3은 COA 자화능이 현저히 저하되었다. 변이주 UV-10은 COA-4 배지로 배양했을 때 친주에 비해 단백질량 30%, Avicel 분해활성 30%, 여지분해활성 20%, salicin 분해활성 50% 증가가 인정되었고, 비록 역가는 낮았지만 glucose 및 cellobiose를 함유하는 배지에서 CMC 및 salicin 분해활성을 구성적으로 생산하였다.

In order to obtain a regulatory mutant strain with high cellulase activity, a newly isolated Penicillium verrculosum, strain F-3 was used as parental strain since it was proved to be an efficient cellulase producer. A number of experiments were conducted to determine the optimum conditions to in-duce mutagenesis and isolate the desirable mutant strains. Out of several restriction compounds tested, 1.5% oxgall was found to be most effective to restrict the colony size by suppressing overgrowth. Derepression of catabolites was employed as a criterion in selecting mutant strains with high cellulase productivity. Production of cellulase by Penicillium venculosum F-3 was suppressed when cultured on the media with more than 1% of glucose or glycerol. It was found that either irradiation with UV light for 19 mins or treatment with nitrosoguanidine at 200$\mu\textrm{g}$/m1 for 60 mins, induced mutagenesis at desired level, when the survival rate of the spore was 0.2% and 48%, respectively. Three mutant strains of F-3, UV-9, UV-10, and NTG-3 that had the highest cellulase productivity were finally selected, based on filter paper degradation rate, size of clearing zone on the screening plate and cellulase activity in the medium containing cellulose powder. When the mutant strains were compared with parental strain F-3, on the KC-M-W medium containing cellulose powder, the filter paper activities of UV-9, UV-10, and NTG-3 were increased by 34%, 55%, and 41%, respectively. However, the assimilation of cellobiose octaacetate by UV-9 or NTG-3 was markedly reduced. When the mutant UV-10 was grown on cellobiose octaacetate medium (CCA-4) in shaking flasks, the cellulase activities of the mutant increased by 20 to 50% compared to the parental strain. Excreation of soluble protein from the mutant also elevated up to 30%. The mutant also constitutively produced both CMCase and $\beta$-glucosidase, though at relatively low level, in the presence of glucose or cellobiose as carbon sources.