Large-Scale Production of Phospholipase D from Streptomyces racemochromogenes and Its Application to Soybean Lecithin Modification
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Phospholipase D (PLD) catalyzes transphosphatidylation, causing inter-conversion of the polar head group of phospholipids and phospholipid hydrolysis. Previously, we cloned PLD103, a PLD with high transphosphatidylation activity, from Streptomyces racemochromogenes strain 10-3. Here, we report the construction of an expression system for the PLD103 gene using Streptomyces lividans as the host bacterium to achieve large-scale production. The phosphatidylcholine (PC) hydrolysis activity of S. lividans transformed with the expression plasmid containing the PLD103 gene was approximately 90-fold higher than that of the original strain. The recombinant PLD103 (rPLD103) found in the supernatant of the transformant culture medium was close to homogeneous. The rPLD103 was indistinguishable from the native enzyme in molecular mass and enzymatic properties. Additionally, rPLD103 had high transphosphatidylation activity on PC as a substrate in a simple aqueous one-phase reaction system and was able to modify the phospholipid content of soybean lecithin. Consequently, the expression system produces a stable supply of PLD, which can then be used in the production of phosphatidyl derivatives from lecithin.
KeywordsPhospholipase D Lecithin Phospholipid Soybean Transphosphatidylation Phospholipid modification Oil manufacture Phosphatidylcholine Phosphatidylserine Phosphatidylglycerol
We thank Kazuaki Hirabayashi, Teppei Kikuchi, and Eri Yamamoto for their technical assistance.
- 8.Suzuki, S., Yamatoya, H., Sakai, M., Kataoka, A., Furushiro, M., & Kudo, S. (2001). Oral administration of soybean lecithin transphosphatidylated phosphatidylserine improves memory impairment in aged rats. The Journal of Nutrition, 131, 2951–2956.Google Scholar
- 12.Yang, S. F., Freer, S., & Benson, A. A. (1967). Transphosphatidylation by phospholipase D. The Journal of Biological Chemistry, 242, 477–484.Google Scholar
- 13.Juneja, L. R., Kazuoka, T., Goto, N., Yamane, T., & Shimizu, S. (1989). Conversion of phosphatidylserine by various phospholipase D in the presence of L- or D-serine. Biochimica et Biophysica Acta, 1003, 277–283.Google Scholar
- 18.Bhosale, S. H., Rao, M. M., & Deshpande, V. V. (1996). Molecular and industrial aspect of glucose isomerase. Microbiological Reviews, 60, 280–300.Google Scholar
- 22.Katz, E., Thompson, C. J., & Hopwood, D. A. (1983). Cloning and expression of the tyrosinase gene from Streptomyces antibioticus in Streptomyces lividans. Journal of General Microbiology, 129, 2703–2714.Google Scholar
- 24.Kieser, T., Bibb, M. J., Buttner, M. J., Chater, K. F., & Hopwood, D. A. (2000). Introduction of DNA into Streptomyces, in practical Streptomyces genetics (pp. 229–252). Norwich: The John Innes Foundation.Google Scholar
- 27.Imamura, S., & Horiuti, Y. (1978). Enzymatic determination of phospholipase D activity with choline oxidase. Journal of Biochemistry, 83, 677–680.Google Scholar
- 29.Ogino, C., Kanemasu, M., Hayashi, Y., Kondo, A., Shimura, N., Tokuyama, S., et al. (2004). Over-expression system for secretory phospholipase D by Streptomyces lividans. Applied Genetics and Molecular Biotechnology, 64, 823–828.Google Scholar
- 30.Carrea, G., D’Arrigo, P., Piergianni, V., Roncaglio, S., Secundo, F., & Servi, S. (1995). Purification and properties of two phospholipases D from Streptomyces sp. Biochimica et Biophysica Acta, 1255, 273–279.Google Scholar
- 32.Ogino, C., Negi, Y., Matsumiya, T., Nakaoka, K., Kondo, A., Kuroda, S., et al. (1999). Purification, characterization, and sequence determination of phospholipase D secreted by Streptoverticillium cinnamoneum. Journal of Biochemistry, 125, 263–269.Google Scholar