Exploration of alternative cell wall lysing enzymes and optimization of conditions for Ganoderma lucidum protoplast isolation

영지 원형질체 분리를 위한 대체 세포벽 분해 효소 탐색과 최적 조건 설정

Recently, active research in Korea and worldwide has begun to focus on gene function and cultivar development using gene editing tools. This research, in addition to studies on edible mushroom, aims to enhance the physical and biochemical characteristics of mushrooms for applications in materials and substance production. For these studies, efficient isolation of protoplasts from the target mushroom is critical. However, several commercial cell wall-lysing enzyme cocktails, including Novozyme234, Glucanex, and Lysing enzymes, have recently been discontinued. In this study, we aimed to identify alternative enzyme systems to replace the discontinued cell wall-lysing enzymes for stable isolation of protoplasts from Ganoderma lucidum. To select an optimal osmotic buffer, enzyme function in 0.6 and 1.2 M Sorbitol, Sucrose, Mannitol, and KCl was assessed. The effect of reaction time was also evaluated. Protoplast isolation efficiency of each alternative enzyme was tested using lysing enzymes from Trichoderma harzianum, Chimax-N, and Yatalase, either individually or in combination. This matrix of studies identified enzymes and optimal conditions that could replace the discontinued lysing enzymes.

버섯의 세포벽 분해를 최적화하기 위한 효소 처리에 따른 원형질체 분리 효율을 분석한 결과는 다음과 같다. 최적 삼투완충액 선발을 위해 Sorbitol, Sucrose, Mannitol, KCl을 각각 0.6M, 1.2M의 두가지 농도로 나누어 비교했을 때 0.6M Sorbitol에서 26.7±3.3×10⁷/mL로 가장 높은 원형질체 분리 효율을 확인하였다. 또한 선발된 삼투완충액과 영지 균사의 반응 시간에 따른 영향을 확인한 결과에서는 4시간 동안 반응했을 때 가장 높은 효율을 보였다. 대체 효소의 경우 endo-, exo-Chitinase의 혼합 효소인 Chimax-N을 사용했을 때 기존의 Lysing enzyme과 Yatalase에 비해 더 높은 분리 효율을 보여 원형질체 분리를 위한 대체 효소로서의 가능성을 확인하였다.