핵전이에 의한 Aspergillus niger와 Penicillium chrysogenum의 잡종에서의 Lipase 고생산균주의 개발

Development of Lipase Hyper-producing Strain from Hybrids between Aspergillus niger and Penicillium chrysogenum by Nuclear Transfer

  • 양영기 (조선대학교 자연과학대학 유전공학과) ;
  • 문명님 (조선대학교 자연과학대학 유전공학과) ;
  • 이윤희 (조선대학교 자연과학대학 유전공학과) ;
  • 임채영 (조선대학교 자연과학대학 유전공학과)
  • 발행 : 1997.03.01

초록

Aspergillus niger와 Penicillium chrysogenum간의 lipase 우수 생성 종간 형질전환체를 획득하고, 유전분석을 통하여 다음과 같은 결과를 얻었다. (1) P. chrysogenum의 영양요구물을 확인하여 본 결과 Tyrosine 요구성임을 알 수 있었다. (2) 핵전이를 위한 원형질체 형성 및 재생 조건에서는 Novozym 234의 농도 1%, 삼투안정제는 0.6M KCl, 효소의 처리 시간은 180분 그리고 최적 pH는 5.8로 나타났다. (3) 핵전이에 의한 형질전환의 빈도는 $1.3{\times}$10^{-3}$$ $-3.8{\times}$10^{-3}$$으로 비교적 낮은 편이었다. (4) 유전적 안정성, conidia의 크기, DNA 함량의 측정 그리고 핵염색의 결과 형질전환체의 핵형은 aneuploid로 추정되었다. (5) 형질전환체의 lipase활성은 모균주와 비교하여 1.4-2.2배 증가하였다.

Intergeneric hybrids between Aspergillus niger and Perricillium ch~y.sop~um(Tyr ), hyperlipolytic enzyne-producing fungi, were obtained by nuclear transfer technique:. Optimal conditions for formation of intergeneric hybrids were investigated. Maximum production of protoplasts were obtainrd by 1% Novozym 234 at $30^{\circ}C$ for 3 hrs and the most effective osmotic stabilizers for the isolation of protoplasts were 0.6 M KC]. Frequencies of hybrid formation by nuclear transfer were $1.3{\times}$10^{-3}$$ $-3.8{\times}$10^{-3}$$. From the chervation of genetic stability, conidial size, DNA content, ;md nuclear stain, it was suggested that their karyotypes are aneuploid. The hybrids showed 1.4-2.2 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.

키워드

참고문헌

  1. Arch. Microbiol. v.105 Condition for induced fusion of fungal protoplasts in polyethylene glycol solution Anne, J.;J.F. Peberdy
  2. Nature v.262 Somatic hy-bridization of Penicillium roquefortii with P. chrysogenum after protoplast fusion Anne, J.;H. Eyssen;P. Desomer
  3. Trans. Br. Mycol. Soc. v.77 Characterization of in-terspecific hybrids between Penicilliuum dhrysogenum and P. roquefortii by isoezyme analysis Anne, J.;J. F. Peberdy
  4. Advances in Protoplast Research Chracterization of hydrolase in protoplasts, mycelium, spores, and abberant tubes of Trichoderma viride Benitez, T.;I. Garcia-Acha;L. Ferenczy;G. L. Farkas (ed.)
  5. J. Gen. Mi-crobiol. v.129 As-pects of genetic interaction in hybrids of Aspergillus ni-dulans and A. rugulosus by protoplast fusion Bradshaw, R.E.;K-U. Lee;J.F. Peberdy
  6. Kor. J. Microbiol. v.19 no.4 Protoplast reversion of Trichoderma koningii Cho, N.T.;H.M. Park;Y.H. Rhee
  7. Production of cellulolytic enzymes by Penicillium ver-ruculosum v.60 Chung, K.C.;K. Kawai;S. Yoshima;Y. Eguchi
  8. Adv. Cargohydr. Chem. Biochem. v.32 Hemicelluases:Their occurrence, purification, properties and mode of ac-tion Dekker, R.F.H;G.N. Richards
  9. Biotechnology and Bioengineering v.115 Bioconversion of hemicellulose: As-pects of hemicellulase production by Trichoderma reesei QM 9414 and enzymic saccharification of hemicellulose Dekker, R.F.H
  10. Technology and Application. Symp. v.6 Enzymatic conversion of cellulosic materials Demain, A.L.
  11. Experientia v.32 Factors affecting high frequency fungal protoplast fusion Ferenczy, L.;F. Kevei;M. Franco;I. Rojik
  12. Curr. Microbiol. v.7 Transfer of nuclei into protoplasts of Saccharomyces cerevisiae Ferenczy, L.;M. Pesti
  13. 6th Inter. Protoplast symposium Current question of gene transfer via protoplast fusion in microorganism Ferenczy, L.
  14. Nature v.206 An improved di-phenylamine method for estimation of deoxyribonucleic acid Giles, K.W.;Myers
  15. Genetic of Industrial microdrganism Recombination studies with Cephalosporium acremonium Hamlyn, P.F.;C. Bull
  16. Kor. J. Microbiol. v.22 The coni-dial protoplast fusion of cellulolytic fungus Trichoderma koningii Hong, S.W.;Y.C. Hah;H. M. Park
  17. Nature v.268 Genetic recombination through protoplast in Streptomyces Hopwood, D.A.;H.M. wright;M.J. Bibb;S.M. Cohen
  18. J. Appl. Bacteriol. v.37 Lipase production and activity as a function of incubation time, pH. and tem-perature of four lipolytic microorganisms Jonsson, U.;B.G. Snygg
  19. Planta v.115 A method for high frequency intergeneric fusion of plant protoplasts Kao, K.N.;M.R. Michayuk
  20. Kor. J. Mycol. v.17 Synergistic effect of substrate on the biosynthesis of cel-luase and xylanase complexs from Aspergillus nidulans Lee, J.A.;J.S. Maeng;P. J. Maeng;Y. H. Rhee
  21. Theor. Appl. Genet v.53 Investigation on the transfer of isolated nuclei into protoplasts Lorz, H.;I. Potrycus
  22. M. S. thesis, Seoul National University Isolation and analysis of intra-and in-terspecific hybids in genus Trichoderma Min, K.R.
  23. J. Biochem. v.71 Production of extracelluar enzymes in mutans isolated from Trichoderma viride Nevalainen, K.M.H.;E.T. Palva
  24. Ph.D.thesis, Seoul National Uniersity Intra-and Interspecific protoplast fusion of celluloytic fungi, Trichoderma koningii and Tri-choderma reesei. Park, H.M.
  25. Trans. Br. Mycol. Soc. v.67 no.1 Factors affecting protoplast release in some filamentous fungi Peberdy, J.F.;C.E. Buckley;D.C. Daltrey;P.M. Moore
  26. Protoplast fusion: A new approach in-terspecific genetic manipulation and breeding in fungi Peberdy, J.F.
  27. Study on interspecific hybrid formation by nuclear transfer in Aspergillus spp. Rho, H.S.
  28. Acta Histochemica v.23 Polyethylene glycol induced membrance fusion in yeast protoplasts Svoboda, A.
  29. Appl. Environ. Microbiol. v.47 Protoplast fusion of Trichoderma reesei using im-mature conidia Toyama, H.;K. Yamaguchi;A. Shinmyo;H. Okada
  30. Method in Microbiol. v.4 Production and use of fungal protoplasts Vilanueva, J. R.;I. Garcia-Acta.;C. Booth (ed.)
  31. Kor. J. Mycol. v.17 Study on formation of Aspergillus nidulans hybrids by pro-toplast fusion and nuclear transfer Yang, Y.K.;Y. Park;Y.H. Rhee;P.J. Maeng