Effects of Low-Shear Modeled Microgravity on the Characterization of Recombinant β-D-Glucuronidase Expressed in Pichia pastoris
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In this study, we used a high-aspect-ratio vessel (HARV), which could model environment of microgravity on ground to investigate for the first time the effects of low-shear modeled microgravity (LSMMG) on the characterization of recombinant β-D-glucuronidase expressed in Pichia pastoris. The β-D-glucuronidase gene (GenBank accession no. EU095019) derived from Penicillium purpurogenum Li-3 encoding β-D-glucuronidase (PGUS) was expressed in P. pastoris GS115 in two different environments of LSMMG and normal gravity (NG). Results manifested that both LSMMG and NG conditions had insignificant effects on temperature and pH activity (optimal temperature and pH were 55 and 5.0 °C, respectively) and characteristic stability of recombinant PGUS. However, the catalytic activity of recombinant PGUS expressed under LSMMG was less affected by metal ions and EDTA as compared with that of NG. Furthermore, K m value of the recombinant PGUS expressed under LSMMG was nearly one fifth of that under NG (1.72 vs. 7.72), whereas catalytic efficiency (k cat/K m) of PGUS expressed under LSMMG (13.55) was 3.7 times higher than that of NG (3.61). The results initially reveal the significant alterations in catalytic properties of recombinant enzyme in response to LSMMG environment and have potential application in bioprocessing and biocatalysis.
KeywordsLow-shear modeled microgravity (LSMMG) Normal gravity (NG) Pichia pastoris Recombinant PGUS
This work is supported by the National “863” High-Tech Project (2008AA12A218-2) and Natural Science Foundation of China (20776017, 20976014) and Natural Science Foundation of Beijing (5072028).
- 6.Fang, A., Pierson, D. L., Mishra, S. K., Koenig, D. W., & Demain, A. L. (1997). Applied and Environmental Microbiology, 63, 4090–4092.Google Scholar
- 10.Hammond, T. G., & Hammond, J. M. (2001). American Journal of Physiology. Renal Physiology, 281, F12–F25.Google Scholar
- 11.Schwarz, R. P., Wolf, D. A., & Trinh, T. (1991). Rotating cell culture vessel. U.S. patent 5,026,650.Google Scholar
- 18.Xiang, L., Qi, F., Dai, D.Z., Li, C., Jiang, Y.D. (2010). Applied Biochemistry and Biotechnology, 162, 654–661.Google Scholar
- 22.Sambrook, J., & Russell, D. W. J. (2001). Molecular cloning: A laboratory manual (3rd ed.). New York: Cold Spring Harbor Laboratory.Google Scholar
- 28.Johanson, K., Allen, P. L., Lewis, F., Cubano, L. A., Hyman, L. E., & Hammond, T. G. (2002). Journal of Applied Physiology, 93, 2171–2180.Google Scholar