One-step purification of alpha-amylase from the cultivation supernatant of recombinant bacillus subtilis by high-speed counter-current chromatography with aqueous polymer two-phase systems.
Purification of alpha-amylase from the cultivation supernatant of recombinant Bacillus subtilis by high-speed counter-current chromatography (HSCCC) in polyethylene glycol (PEG) 4000-inorganic salt aqueous polymer two-phase systems was studied. The effects of sodium chloride concentration on the partition coefficients of alpha-amylase and total protein were respectively tested in PEG4000-phosphate and PEG4000-citrate aqueous polymer two-phase systems to find the proper range of sodium chloride concentration for the HSCCC purification of alpha-amylase. Alpha-amylase was purified from the cultivation supernatant by HSCCC in PEG4000-phosphate system containing 2% (w/w) sodium chloride, yet with considerable loss of activity. PEG4000-citrate aqueous polymer two-phase system containing 2% (w/w) sodium chloride and supplemented with 0.56% (w/w) CaCl2 as protective agent was then successfully applied to purify alpha-amylase from cultivation supernatant by HSCCC to homogeneity and significantly increased the recovery of alpha-amylase activity from around 30 to 73.1%.
Zhi
Wenbo
W
National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, The Chinese Academy of Sciences, Beijing 100080, China. zhiwenbo@163.com
Deng
Qiuyun
Q
Song
Jiangnan
J
Gu
Ming
M
Ouyang
Fan
F
D001412:Bacillus subtilis / Q000201:enzymology*; D003377:Countercurrent Distribution / Q000379:methods*; D003470:Culture Media; D004591:Electrophoresis, Polyacrylamide Gel; D011108:Polymers / Q000737:chemistry*; D011995:Recombination, Genetic; D014867:Water / Q000737:chemistry; D000516:alpha-Amylases / Q000302:isolation & purification*
D003470:Culture Media; D011108:Polymers; D014867:Water; D000516:alpha-Amylases