초록

Aspergillus niger와 Penicillium notatum간의 lipase 우수 생성 종간 형질전환체를 획득하고, 유전분석과 정제를 통하여 다음과 같은 결과를 얻었다. P. notatum의 영양요구물을 확인하여 본 결과 Tyrosine 요구성임을 알 수 있었다. 핵전이를 위한 원형질체 형성 및 재생 조건에서는 Novozym 234의 농도 1%, 삼투안정제는 0.6M KCl, 효소의 처리시간은 180분 그리고 최적 pH는 5.8로 나타났다. 핵전이에 의한 형질전환의 빈도는 $1.3{\times}10^{-4}{\sim}3.8{\times}10^{-4}$으로 비교적 낮은 편이었다. 유전적 안정성, conidia의 크기, DNA 함량의 측정 그리고 핵염색의 결과 형질전환체의 핵형은 aneuploid로 추정되었다. 형질전환체의 lipase 활성은 모균주와 비교하여 $1.2{\sim}1.7$배 증가하였다.

Abstract

Interspecific hybrids between Aspergillus niger and Penicillium notatum (Tyr-), hyperlipolytic enzyme-producing fungi, were obtained by nuclear transfer technique. Optimal conditions for formation of intergeneric hybrids were investigated. Maximum production of protoplasts was obtained by 1% Novozyme 234 at $30^{\circ}C$ for 3 hrs and the most effective osmotic stabilizers for the isolation of protoplasts were 0.6 M KCl. Frequencies of hybrid formation by nuclear transfer were $3.8{\times}10^{-3}{\sim}1.3{\times}10^{-3}$. From the observation of genetic stability, conidial size, DNA content, and nuclear stain, it was suggested that their karyotypes are aneuploid. The hybrids showed $1.2{\sim}1.7$ fold higher lipase activities than parental strains. It was strongly supported by results of this study that nuclear transfer technique is much more efficient in the formation of intergeneric hybrids than protoplast fusion and is very useful for the improvement of strains.

주제어

#Aspergillus niger   #Penicillium notatum   #Nuclear transfer   #Lipase  

저자의 다른 논문

참고문헌 (31)

1. Condition for induced fusion of fungal protoplasts in polyethylene glycol solution , Anne, J.;Peberdy, J.F. , Arch. Microbiol. / v.105,pp.201-205, 1975
   
2. Somatic hybridization of Penicillium roquefortii with P. chrysogenum after protoplast fusion , Anne;Eyssen, J.H.;Desomer, P. , Nature / v.262,pp.719-721, 1976
   
3. Characterization of interspecific hybrids between Penicilliuum chrysogenum and P. roquefortii by isoenzyme analysis , Anne, J.;Peberdy, J.F. , Trans. Br. Mycol. Soc. / v.77,pp.401-408, 1981
   
4. Chracterization of hydrolase in protoplasts, mycelium, spores, and abberant tubes of Trichoderma uiride , Benitez, T.;Garcia-Acha, I.;Ferenczy, L.(ed.);Farkas, G.L.(ed.) , / v.,pp.199-204, 1988
   
5. Aspects of genetic interaction in hybrids of Aspergillus nidulans and A. rugulosus by protoplast fusion , Bradshaw, R.E.;Lee, K.U.;Peberdy, J.F. , J. Gen. Microbiol. / v.129,pp.3525-3533, 1983
   
6. Protoplast reversion of Trichoderma koningii , Cho, N.T.;Park, H.M.;Rhee, Y.H. , Kor. J. Microbiol. / v.19,pp.192-198, 1982
   
7. Production of cellulolytic enzymes by Penicillium uerruculosum , Chung, K.C.;Kawai, K.;Yoshima, S.;Eguchi, Y. , Hakkokogaku / v.60,pp.355-357, 1982
   
8. Hemicellulases: Their occurrence, purification, properties and mode of action , Dekker, R.F.H.;Richards, G.N. , Adu. Carbohydr. Chem. Biochem. / v.32,pp.276-352, 1976
   
9. Bioconversion of hemicellulose: Aspects of hemicellulase production by Trichoderma reesei QM 9414 and enzymic saccharification of hemicellulose , Dekker, R.F.H. , Biotechnology and Bioengineering / v.115,pp.1127-1146, 1983
   
10. Enzymatic conversion of cellulosic materials , Demain, A.L. , Technology and Application. Symp. / v.6,pp.79-81, 1976
    
11. Factors affecting high frequency fungal protoplast fusion , Ferenczy, L.;Kevei, F.;Franco, M.;Rojik, I. , Experientia / v.32,pp.1156-1158, 1976
    
12. Transfer of nuclei into protoplasts of Saccharomyces cerevisiae , Ferenczy, L.;Pesti, M. , Curr. Microbiol / v.7,pp.157-160, 1982
    
13. Current question of gene transfer via protoplast fusion in microorganism , Ferenczy, L. , 6th Inter. Protoplast symposium / v.,pp.137-142, 1983
    
14. An improved diphenylamine method for estimation of deoxyribonucleic acid , Giles, K.W.;Myers , Nature / v.206,pp.93, 1965
    
15. Recombination studies with Cephalosporium acremonium;Genetic of Industrial microdrganism , Hamlyn, P.F.;Bull, C. , / v.,pp.185-191, 1979
    
16. The conidial protoplast fusion of cellulolytic fungus, Trichoderma koningii , Hong, S.W.;Hah, Y.C.;Park, H.M. , Kor. J. Microbiol / v.22,pp.207-213, 1984
    
17. Genetic recombination through protoplast in Streptomyces , Hopwood, D.A.;Wright, H.M.;Bibb, M.J.;Cohen, S.M. , Nature / v.268,pp.171-174, 1977
    
18. Lipase production and activity as a function of incubation time, pH. and temperature of four lipolytic microorganisms , Jonsson, U.;Snygg, B.G. , J. Appl. Bacteriol / v.37,pp.571-581, 1974
    
19. A method for high frequency intergeneric fusion of plant protoplasts , Kao, K.N.;Michayuk, M.R. , Planta / v.115,pp.355-367, 1974
    
20. Synergistic effect of substrate on the biosynthesis of cellulase and xylanase complexes from Aspergillus nidulans , Lee, J.A.;Maeng, J.S.;Maeng, P.J.;Rhee, Y.H. , Kor. J. Mycol / v.17,pp.57-65, 1989
    
21. Investigation on the transfer of isolated nuclei into protoplasts , Lorz, H.;Potrycus, I. , Theor. Appl. Genet / v.53,pp.251-256, 1978
    
22. Isolation and analysis of intraand interspecific hybrids in genus Trichoderma , Min, K.R. , / v.,pp., 1987
    
23. Production of extracellular enzymes in mutants isolated from Trichoderma viride , Nevalainen, K.M.H.;Palva, E.T. , J. Biochem / v.71,pp.999-1007, 1978
    
24. Intra- and Interspecific protoplast fusion of cellulolytic fungi, Trichoderma koningii and Trichoderma reesei , Park, H.M. , / v.,pp., 1985
    
25. Factors affecting protoplast release in some filamentous fungi , Peberdy, J.F.;Buckley, C.E.;Daltrey, D.C.;Moore, P.M. , Trans. Br. Mycol. Soc. / v.67,pp.23-26, 1976
    
26. Protoplast fusion: A new approch interspecific genetic manipulation and breeding in fungi , Peberdy, J.F. , / v.,pp.63-71, 1980
    
27. Study on interspecific hybrid formation by nuclear transfer in Aspergillus spp. Department biology , Rho, H.S. , / v.,pp., 1991
    
28. Polyethylene glycol induced membrane fusion in yeast protoplasts , Svoboda, A. , Acta Histochemica / v.23,pp.211-217, 1981
    
29. Protoplast fusion of Trichoderma reesei using immature conidia , Toyama, H.;Yamaguchi, K.;Shinmyo, A.;Okada, H. , Appl. Environ. Microbiol / v.47,pp.363-368, 1984
    
30. Production and use of fungal protoplasts , Villanueva, J.R.;Garcia-Acta, I.;Booth, C.(ed.) , Method in Microbiol / v.4,pp.665-718, 1971
    
31. Study on formation of Aspergillus nidulans hybrids by protoplast fusion and nuclear transfer , Yang, Y.K.;Park, Y.;Rhee, Y.H.;Maeng, P.J. , Kor. J. Mycol / v.17,pp.154-160, 1989
    

이 논문을 인용한 문헌 (0)

1. 이 논문을 인용한 문헌 없음