Extracellular release of recombinant alpha-amylase from Escherichia coli using pulsed electric field.
It is difficult for Escherichia coli to secrete products such as recombinant enzymes, because the Gram-negative bacterium has a double membrane structure and so some of the products are accumulated in a periplasmic space. In this study, we demonstrated that recombinant alpha-amylase can be released from recombinant E. coli HB101/pHI301A during cultivation by applying a pulsed electric field (PEF). When a PEF (12 kV, 2 Hz) was applied for 30 min with an interval of 30 min from the point of OD660=0.7, the amount of released alpha-amylase was about 30% of the total amount of alpha-amylase produced in the cells. As a result of SDS-PAGE and activity staining analyses, it was confirmed that the released proteins were not all of the intracellular proteins, and the alpha-amylase, which was identical with intracellular alpha-amylase, was released by applied PEF cultivation. PEF treatment could be useful for easy release of periplasmic protein with selectivity.
Department of Biological and Chemical Engineering, Faculty of Engineering, Gunma University, 1-5-1 Tenjin-cho, Kiryu, Gunma, 376-8515, Japan. tohshima@bce.gunma-u.ac.jp
Satoshi
S
Shiina
Takayuki
T
Ohshima
Masayuki
M
Sato
D019149:Bioreactors / Q000382:microbiology; D018929:Cell Culture Techniques / Q000379:methods; D002458:Cell Fractionation / Q000379:methods*; D004058:Diffusion; D004307:Dose-Response Relationship, Radiation; D004574:Electromagnetic Fields; D018274:Electroporation / Q000379:methods*; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics / Q000528:radiation effects*; D045604:Extracellular Fluid / Q000737:chemistry*; D015202:Protein Engineering / Q000379:methods; D011829:Radiation Dosage; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D000516:alpha-Amylases / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification
D011994:Recombinant Proteins; D000516:alpha-Amylases