Characteristics of Protoplast Induction by Autolytic Enzyme of Rhizopus oryzae

Autolytic Enzyme을 이용한 Rhizopus oryzae의 원형질체 생성과 그 특성에 관한 연구

The degree of autolysis and lytic enzyme production in the culture filtrate of Rhizopus oryzae was investigated. The formation of protoplast by using autolytic enzymes from Rh. oryzae was also attempted. Protoplasts were liberated from Rh. oryzae mycelium by lytic enzymes present in autolytic-phase culture filtrate. Maximum release of chitosanase and proteolytic enzyme into culture filtrate during autolysis was corresponded to maximum protoplast-liberating activity. High yields of protoplasts were obtained from 10 hr-age of Rh. oryzae mycelium with 0.5 M mannitol as osmotic stabilizer. The optimum temperature and pH for mycelium digestion were $25{\sim}30^{\circ}C$ and $6.0{\sim}6.5$ respectively. The mycelium of the 18 hours cultures were treated with autolytic enzyme in same volume of osmotic stabilizer at $30^{\circ}C$ for 5 hours and then it was confirmed by scanning electoron microscope that protoplast were produced beside the digesting cell wall of the fungi.

Rhizorus oryzae의 생장 및 autolysis에 따른 여러가지 autolytic enzymes의 활성도 변화와 이 autolytic enzyme system을 이용한 원형질체 생성에 관하여 조사하였다. Autolytic·phase의 culture filtrate 내에 함유된 autolytic enzyme은 Rh. oryzae 균사체로부터의 원형질체 생성에 효율적인 세포벽 분해효소로 작용하였으며 Autolytic enzyme중 원형질체 생성은 proteolytic 및 chitosanase activity와 가장 긴밀하게 관련되어 있었다. 이와같은 autolytic enzyme을 이용한 원형질체 생성은 10시간 배양한 균사체에 0.5M mannitol을 사용하였을 때 최고의 수율을 보였으며 원형질 생성의 최적온도 및 pH는 각각 $25{\sim}30^{\circ}C$ 및 $6.0{\sim}6.5$로 나타났다 한편 18시간 배양된 균사체를 osmotic stabilizer와 aut-olytic enzyme을 1 : 1이 되도록 처리하고 5시간 동안 $30^{\circ}C$에서 incubation하여 얻은 원형질체를 주사전자현미경으로 조사한 결과 효소에 의하여 가수분해되는 세포벽 주변으로부터 원형질체가 형성되는 것을 확인하였다.