The objective of present study was to assess the possibility of introducing enzymatic technique for the milder recovery of intracellular lipids from R. gracilis.
A F5 fungus producing cell wall lytic enzyme was isolated from soil. This lytic F5 fungus was identified as the species of Aspergillus fumigatus group. Crude lytic enzyme of F5 fungus was of inducible exo-enzyme, and it was able to promote the lysis of R.gracilis intact cells in fattening phase and growing phase. R. gracilis intact cells in growing phase were more susceptible to lysis than those in fattening phase.
Lytic enzyme system was composed of, at least, lytic enzyme and protease, which acted cooperatively in the lysis of intact cells. Lytic enzyme and protease were separated on Bio-Gel P-60 column chromatography. Lytic enzyme was not able to hydrolyze β-1, 3- and β-1,6-glucan which were known to the main types of bonds found in general yeast cell wall, such as Ascomycetous yeast. The specificity of lytic enzyme remained obscure. This lytic enzyme alone was sufficient to hydrolyze the fractionated cell wall of R.gracilis(alkali-insoluble-residues). This result well supported the hypothetical structure of yeast cell wall, which suggested the presence of mannoprotein complexes on the outer layer of yeast glucan.
Lytic enzyme had its pH optimum between pH 4-4.5 and had its temperature optimum 50℃. And, it had broad pH stability between 3.5 and 7.5. Heating of this enzyme for 20 min at 50℃ resulted in 80% loss in enzyme activity. When the protease was assayed with azocasein, its pH optimum and temperature optimum were pH 8.0-9.5,50℃, respectively.
균체내 유지 생성효모로 알려져온 Rhodotorula gracilis 로 부터 유지를 온화한 조건에서 추출하기 위하여, 세포벽의 효소에 의한 제거에 대해 연구 하였다.
R. gracilis 세포벽 분해 효소를 생산하는 F5 곰팡이를 토양으로 부터 분리하여 동정한 결과 Aspergillus sp. 였다. F5 곰팡이로 부터 생산되는 효소는 균체의 효소 였으며 유도 효소 였다. 이 효소는 R. gracilis 균체가 성장단계에 있거나 유지 생성단계에 있거나 세포벽을 분해하여 원형질체로 전환 시킬수 있었다.
이 세포벽 분해효소는 적어도 protease 와 lytic enzyme 으로 구성되어 있으며, 두가지 효소가 R. gracilis 생세포를 파괴 하는데 상승적으로 작용 하였다. 이 두 효소는 Bio-Gel P-60 column chromatography 에 의해 분리될수 있었으며, lytic enzyme 은 Ascomycetous 효소 등의 세포벽에 주로 존재하는 β-1,3-,β-1,6-glucan 에는 작용치 않았다. 또한 이 lytic enzyme 은 R. gracilis 세포의 알카리 불용성 획분 (alkali insoluble residues) 에 protease 의 도움없이 작용할수 있었다. 이 효소의 최적 pH 는 4-4.5, 최적온도는 50℃였으며, pH 3.5 - 7.5 사이에서 안정성을 보였다. 또한 protease를 azocasein 으로 역가 측정하였을때 최적 pH는 8 - 9.5, 최적온도는 50 - 60℃ 였다.
