Abstract
The effects of key parameters on the preparation and regeneration of protoplast from the β-carotene-producing fungus Blakeslea trispora were discussed in this paper, including the combination of various enzymes, mycelial age, digesting time and temperature, pH value, osmotic stabilizers, pretreatment, culture medium and culture method. Under the condition of mixed enzymes in osmotic stabilizer (0.6 M NaCl) combined with 2% lysozyme, 3% cellulase and 3% snailase, the highest protoplast yield, as high as 7.48×106 protoplasts/mL, was obtained when mycelial age was 60 h at pH 5.0–6.0 with digesting for 14–16 h at 28 °C. After purification of the obtained protoplasts, they were regenerated in PDA regenerative medium using bilayer plate culture method. To validate the usability of the protoplasts, a novel plasmid with green fluorescent protein (GFP) was used in transformation for easy visual observation. The results showed that the protoplasts prepared by the optimized method were active and applicable in further gene manipulation experiments.
Article PDF
Similar content being viewed by others
Avoid common mistakes on your manuscript.
References
B.K. Kim, P. K. Park, H. J. Chae and E.Y. Kim, Korean J. Chem. Eng., 21, 689 (2004).
O. Austa, N. Ale-Aghaa and Z.G. Li, Food Chem. Toxico., 41, 1399 (2003).
A. Rahman and R. S. Parker, Nutr. Res., 21, 581 (2001).
M. Stacewicz-Sapuntzakis and P. E. Bowen, Biochimica Biophysica Acta., 1740, 202 (2005).
K. Wertz, U. Siler and R. Goralczyk, Arch Biochem Biophys., 430, 127 (2004).
S. Yilmaz, A. Atessahin and E. Sahna, Toxicology, 218, 164 (2006).
R. M. McClain and J. Bausch. Regul.Toxico. Pharm., 37, 274 (2003).
G. Vasapollo, L. Longo and L. Rescio, J. Supercrit. Fluid, 29, 87 (2004).
F. Xu, Q. P. Yuan and Y. Zhu, Process biochem., Article in Press.
Z.M. Ou, J. P. Wu, L. R. Yang and P. L. Cen, Korean J. Chem. Eng., 25, 124 (2008).
J.H. Seo, H. X. Li, M. J. Kim and S. J. Kim, Korean J. Chem. Eng., 24, 800 (2007).
D.H. Kang, E. J. Jeh, J.W. Seo, B.H. Chun and B.K. Hur, Korean J. Chem. Eng., 24, 651 (2007).
H. J. Cha, K. R. Kim, B. H. Hwang, D. H. Ahn and Y. J. Yoo, Korean J. Chem. Eng., 24, 812 (2007).
H. I. Lee, B. S. Yoo, M.A. Yoo and S.Y. Byun, Korean J. Chem. Eng., 24, 655 (2007).
Y. G. Li, J.M. Xing, W. L. Li, X. C. Xiong, X. Li and H. Z. Liu, Korean J. Chem. Eng., 24, 781 (2007).
S.M. Lee, W. J. Chang, A. R. Choi and Y. M. Koo, Korean J. Chem. Eng., 22, 687 (2005).
J.Y. Jung, T. Khan, J.K. Park and H.N. Chang, Korean J. Chem. Eng., 24, 265 (2007).
J.Y. Jung, T. Khan, J.K. Park and H.N. Chang, Korean J. Chem. Eng., 24, 265 (2007).
A. Arpornwichanop and N. Shomchoam, Korean J. Chem. Eng., 24, 11 (2007).
J.K. Park and K. D. Lee, Korean J. Chem. Eng., 18, 363 (2001).
J. K. Park, J. Y. Jung and Y. H. Park, Biotechnol. Lett., 25, 2055 (2003).
K. S. Seo, H.N. Chang, J.K. Park and K.H. Choo, Appl. Microbiol. Biotechnol., 76, 951 (2007).
D.B. Choi, H.G. Nam and W. S. Cha, Korean J. Chem. Eng., 23, 241 (2006).
F. Mantzouridou, T. Roukas and P. Kotzekidou, Biochem. Eng. J., 10, 123 (2002).
J. H. Lee and Y. T. Kim, Gene, 370, 86 (2006).
Y. L. Cheng and R. R. Bélanger, FEMS Microbiol. Lett., 190, 287 (2000).
V.D. Mukund and V. C. Manisha, Microbiol. Res., 157, 29 (2004).
D. Q. R. María, M. R. V. Rosa and G. Victoriano, Fungal Genet. Biol., 42, 141 (2005).
J.Y. Yu, S. L. DeRuiter and D. L. Turner, Proc. Natl. Acad. Sci., 99, 6047 (2002)
L. Manczinger, Z. S. Antal and L. Ferenczy, FEMS Microbiol. Lett., 130, 59 (1995).
B.G. Turgeon, R.C. Garber and O. C. Yoder, Mol. Cell Biol., 7, 3297 (1987).
Z.G. Zhang, The isolation and identification of protoplast, Hunan Sci. Technol. Publications, Hunan (2003).
F. Sergi, R. Daniel and S. Joan, Curr. Microbiol., 12, 301 (1985).
X. Y. Chen and R. Hampp, Curr. Microbiol., 26, 307 (1993).
A. S. Sonnenberg, J.G. Wessels and L. J.V. Griensven, Curr. Microbiol., 17, 285 (1988).
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Li, Y., Yuan, Q. & Du, X. Protoplast from β-carotene-producing fungus Blakeslea trispora: Preparation, regeneration and validation. Korean J. Chem. Eng. 25, 1416–1421 (2008). https://doi.org/10.1007/s11814-008-0232-x
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11814-008-0232-x