Rhizopus nigricans로부터 원형질체의 분리

Isolation of Protoplasts from Rhizopus nigricans

Rhizopus nigricans의 포자와 균사체로부터 원형질체 생성을 위한 최적 조건을 조사하였다. 5% 2-deoxy-D-glucose가 첨가된 액체 배지에서 14시간 배양시킨 균사체보다 동일배지에서 4시간 배양하여 팽창시킨 포자로부터 더 많은 원형질체가 생성되었으며, Novozym 234(2%)를 단독으로 처리하였을 때보다는 5000 unit/ml 의 ${\beta}-glucuronidase$와 혼합하였을 때 원형질체의 생성율이 더 높았다. 삼투 안정제로 0.6 M $MgSO_4$를 사용한 복합 효소 용액(pH 6.0)에 2시간 처리하였을 때 팽창된 포자로부터 단위ml당 $8.0{\times}10^6$의 원형질체를 분리할 수 있었다.

Conditions for isolation of protoplasts from spores and mycelia of Rhizopus nigricans were studied. Larger amount of protoplasts was obtained from swollen spores in liquid medium contained with 5% of 2-deoxy-D-glucose for 4 hours than from mycelia. Enzyme mixture of Novozym 234(2%) and ${\beta}-glucuronidase(5000\;unit/ml)$ was most effective for the isolation of protoplasts from swollen spores and from mycelia. The solution of 0.6 M $MgSO_4$ or mannitol and pH 6.0 showed good results as the osmotic stabilizer and the optimal condition of pH of the enzyme solution for the isolation of protoplast from the swollen spores, respectively. At this condition, $8.0{\times}10^6\;cells/ml$ of protoplasts was obtained from swollen spores by digestion with lytic enzyme mixture for 2 hours.