One-step purification of α-amylase from the cultivation supernatant of recombinant Bacillus subtilis by high-speed counter-current chromatography with aqueous polymer two-phase systems

Wenbo Zhi, Qiuyun Deng, Jiangnan Song, Ming Gu, Fan Ouyang

Research output: Contribution to journalArticle

13 Scopus citations

Abstract

Purification of α-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 α-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 α-amylase. α-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 α-amylase from cultivation supernatant by HSCCC to homogeneity and significantly increased the recovery of α-amylase activity from around 30 to 73.1%.

Original languageEnglish (US)
Pages (from-to)215-219
Number of pages5
JournalJournal of Chromatography A
Volume1070
Issue number1-2
DOIs
StatePublished - Apr 8 2005
Externally publishedYes

Keywords

  • Aqueous polymer two-phase system
  • High-speed counter-current chromatography
  • Purification
  • α-Amylase

ASJC Scopus subject areas

  • Analytical Chemistry
  • Biochemistry
  • Organic Chemistry

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