• ポスター番号： 1P-133
  • 日時： 9月9日　 13:00～14:00
  • 演題： Bacterial surface display of functional enzyme using Bacillus subtilis spore
  • 発表者： June-Hyung Kim（Dept. Chem. Eng., Dong-A Univ.）

講演要旨

Bacterial surface display technology has been acknowledged as a useful tool in various fields of biotechnological application such as in vitro evolved enzyme itself, enzymes or proteins involved in whole cell bioconversion, bioremediation, whole cell adsorption and live vaccine production. For the functional bacterial surface display of active enzyme of multimeric form, which is generally impossible due to molecular assembly of the monomer subunit subsequent to the secretion of displayed target protein outside the cell, a new surface display system based on B. subtilis spore should be developed.
Using cotE and cotG of B. subtilis as anchoring motives, beta-galactosidase, which is active in tetrameric form, was functionally displayed on the surface of B. subtilis spore. The surface localization of beta-galactosidase was verified by Miller assay of purified spore, protease accessibility test of purified spore, and flow cytometric analysis of spore expressing beta-galactosidase. While B. subtilis spore wall integrity, examined by lysozyme and heat treatments, was affected by the incorporation of CotE-LacZ fusion protein, it was not affected by the incorporation of CotG-lacZ fusion. Heat stability of displayed protein was similar with that of free enzyme.
In this study, we developed a general method for the efficient surface display system for the tetrameric protein using B. subtilis spore. Using the same anchoring motif (cotG), we could successfully surface displayed another tetrameric protein (streptavidin) and dimeric protein (w-transaminase) and GFP. One more advantage of this system is the size of functionally displayed beta-galactosidase (4*116 kDa = 464 kDa), of which size was not possible to be displayed with current bacterial surface display system. This spore display system also overcame the toxicity caused by fusion protein accumulation on the transport machinery or titration of export component.