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Optimization of the pilot-scale production of an ice-binding protein by fed-batch culture of Pichia pastoris

  • Biotechnological products and process engineering
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Abstract

Ice-binding proteins (IBPs) can bind to the ice crystal and inhibit its growth. Because this property of IBPs can increase the freeze–thaw survival of cells, IBPs have attracted the attention from industries for their potential use in biotechnological applications. However, their use was largely hampered by the lack of the large-scale recombinant production system. In this study, the codon-optimized IBP from Leucosporidium sp. (LeIBP) was constructed and subjected to high-level expression in methylotrophic Pichia pastoris system. In a laboratory-scale fermentation (7 L), the optimal induction temperature and pH were determined to be 25 °C and 6.0, respectively. Further, employing glycerol fed-batch phase prior to methanol induction phase enhanced the production of recombinant LelBP (rLeIBP) by ∼100 mg/l. The total amount of secreted proteins at these conditions (25 °C, pH 6.0, and glycerol fed-batch phase) was ∼443 mg/l, 60 % of which was rLeIBP, yielding ∼272 mg/l. In the pilot-scale fermentation (700 L) under the same conditions, the yield of rLeIBP was 300 mg/l. To our best knowledge, this result reports the highest production yield of the recombinant IBP. More importantly, the rLeIBP secreted into culture media was stable and active for 6 days of fermentation. The thermal hysteresis (TH) activity of rLeIBP was about 0.42 °C, which is almost the same to those reported previously. The availability of large quantities of rLeIBP may accelerate further application studies.

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Acknowledgments

This work was supported by the National Agenda Project from The Korea Research Council of Fundamental Science & Technology (KRCF; grant no. PG12010).

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Author notes

  1. Jong Chan Park

    Present address: Department of Plant Molecular Systems Biotechnology & Crop Biotech Institute, Kyung Hee University, Yongin, 446-701, South Korea

Authors and Affiliations

  1. Division of Polar Life Sciences, Korea Polar Research Institute, Incheon, 406-840, South Korea

    Jun Hyuck Lee, Sung Gu Lee, Hackwon Do, Jong Chan Park, Eunjung Kim, Yong-Hoe Choe, Se Jong Han & Hak Jun Kim

  2. Department of Polar Sciences, University of Science and Technology, Incheon, 406-840, South Korea

    Jun Hyuck Lee, Sung Gu Lee, Hackwon Do & Hak Jun Kim

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  1. Jun Hyuck Lee
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Correspondence to Se Jong Han or Hak Jun Kim.

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J. H. Lee and S. G. Lee contributed equally to this manuscript.

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Lee, J.H., Lee, S.G., Do, H. et al. Optimization of the pilot-scale production of an ice-binding protein by fed-batch culture of Pichia pastoris . Appl Microbiol Biotechnol 97, 3383–3393 (2013). https://doi.org/10.1007/s00253-012-4594-y

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