Isolation of polyhydroxyalkanoate from hydrolyzed cells of Bacillus flexus using aqueous two-phase system containing polyethylene glycol and phosphate

M. S. Divyashree, T. R. Shamala, N. K. Rastogi

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18 Citations (Scopus)

Abstract

Main objective of present work was to isolate polyhydroxyalkanoate (PHA) from cell lysate of Bacillus flexus by aqueous-aqueous two-phase system (ATPS). Selected ATPS having polyethylene glycol (12%, w/v) and potassium phosphate (9.7%, pH 8.0) containing cell lysate obtained by sonication or hypochlorite treatment of B. flexus biomass (1 g%, dry weight), was held at 28°C for 30 min, which partitioned PHA into top PEG phase and residual cell materials into bottom phase. For enzymatic cell hydrolysis, Microbispora sp. culture filtrate having protease (3 U/mL) was mixed with B. flexus biomass and ATPS, incubated at 37°C for 2 h prior to phase separation. PHA recovered by centrifugation was 19∼51% of cell dry weight, depending on the mode of cell disruption. Protease was recovered along with PHA in the PEG phase and showed 7 fold increase in activity. PHA was characterized by GC, FTIR, and 1H NMR. Results indicated that ATPS can be used for the isolation of PHA from hydrolyzed bacterial cells and purified protease can be recovered as a byproduct, in a single defined experiment. Results have indicated that ATPS can be successfully employed as a non-organic solvent method for the isolation of PHA.

Original languageEnglish
Pages (from-to)482-489
Number of pages8
JournalBiotechnology and Bioprocess Engineering
Volume14
Issue number4
DOIs
Publication statusPublished - 09-10-2009

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Polyhydroxyalkanoates
Bacilli
Bacillus
Polyethylene glycols
Phosphates
Biomass
Sonication
Centrifugation
Peptide Hydrolases
Phase separation
Byproducts
Potassium
Hydrolysis
Nuclear magnetic resonance
Hypochlorous Acid
Weights and Measures
Fourier Transform Infrared Spectroscopy
Experiments

All Science Journal Classification (ASJC) codes

  • Biotechnology
  • Bioengineering
  • Applied Microbiology and Biotechnology
  • Biomedical Engineering

Cite this

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title = "Isolation of polyhydroxyalkanoate from hydrolyzed cells of Bacillus flexus using aqueous two-phase system containing polyethylene glycol and phosphate",
abstract = "Main objective of present work was to isolate polyhydroxyalkanoate (PHA) from cell lysate of Bacillus flexus by aqueous-aqueous two-phase system (ATPS). Selected ATPS having polyethylene glycol (12{\%}, w/v) and potassium phosphate (9.7{\%}, pH 8.0) containing cell lysate obtained by sonication or hypochlorite treatment of B. flexus biomass (1 g{\%}, dry weight), was held at 28°C for 30 min, which partitioned PHA into top PEG phase and residual cell materials into bottom phase. For enzymatic cell hydrolysis, Microbispora sp. culture filtrate having protease (3 U/mL) was mixed with B. flexus biomass and ATPS, incubated at 37°C for 2 h prior to phase separation. PHA recovered by centrifugation was 19∼51{\%} of cell dry weight, depending on the mode of cell disruption. Protease was recovered along with PHA in the PEG phase and showed 7 fold increase in activity. PHA was characterized by GC, FTIR, and 1H NMR. Results indicated that ATPS can be used for the isolation of PHA from hydrolyzed bacterial cells and purified protease can be recovered as a byproduct, in a single defined experiment. Results have indicated that ATPS can be successfully employed as a non-organic solvent method for the isolation of PHA.",
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AU - Shamala, T. R.

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AB - Main objective of present work was to isolate polyhydroxyalkanoate (PHA) from cell lysate of Bacillus flexus by aqueous-aqueous two-phase system (ATPS). Selected ATPS having polyethylene glycol (12%, w/v) and potassium phosphate (9.7%, pH 8.0) containing cell lysate obtained by sonication or hypochlorite treatment of B. flexus biomass (1 g%, dry weight), was held at 28°C for 30 min, which partitioned PHA into top PEG phase and residual cell materials into bottom phase. For enzymatic cell hydrolysis, Microbispora sp. culture filtrate having protease (3 U/mL) was mixed with B. flexus biomass and ATPS, incubated at 37°C for 2 h prior to phase separation. PHA recovered by centrifugation was 19∼51% of cell dry weight, depending on the mode of cell disruption. Protease was recovered along with PHA in the PEG phase and showed 7 fold increase in activity. PHA was characterized by GC, FTIR, and 1H NMR. Results indicated that ATPS can be used for the isolation of PHA from hydrolyzed bacterial cells and purified protease can be recovered as a byproduct, in a single defined experiment. Results have indicated that ATPS can be successfully employed as a non-organic solvent method for the isolation of PHA.

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