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Received 10 May 2002; received in revised form 5 August 2002; accepted 9 August 2002
Abstract
An /5.0 kb Sau3A I genomic DNA fragment from Streptomyces aureofaciens NRRL 2209 was cloned in a plasmid vector and
introduced into Escherichia coli . The recombinant E. coli accumulated polyhydroxyalkanoates (PHAs) as cytoplasmic inclusions.
The accumulated PHA was identified as the isotactic homopolymer of PHB with a molecular weight of 2.85 /105. Purified PHB
granules were spherical with an average size of 1.1 mm and of stable configuration. DSC thermogram suggested high crystalline
nature of the polymer. Maximum thermal degradation of the biopolymer occurred between 250 and 340 8C. Recombinant E. coli
cells preferentially utilized glycerol as the carbon source and accumulated 25 /28 times more PHB than the native S. aureofaciens .
# 2002 Elsevier Science B.V. All rights reserved.
for the first time, the characterization data of the blotted dry. Before examination, the slide was remois-
biopolymer PHB (poly-3-hydroxybutyrate) synthesized tened with water and a cover slip was placed on the
by recombinant E. coli harboring an approximately 5.0 smear [22]. The slides were observed under the oil
kb genomic DNA fragment from S. aureofaciens NRRL immersion lens of a Leica fluorescent microscope at an
2209. Also, we report 25 /28 times higher PHB accu- excitation wavelength of 480 nm.
mulation by the recombinant E. coli than the native
organism S. aureofaciens . 2.4. FTIR analysis
Heat fixed bacterial smear was stained with 1.0% Thermogravimetric change of PHA sample was
aqueous solution of Nile blue A at 55 8C for 10 min. analyzed using Rheometric TG analyser (Rheometric
The slide was washed with tap water to remove excess Scientific, USA). The analysis was carried out under
stain and then with 8% aqueous acetic acid for 1 min. nitrogen flow rate of 15 ml/min with a scanning rate of
The stained smear was again washed with water and 10 8C/min.
T.V.N. Ramachander et al. / International Journal of Biological Macromolecules 31 (2002) 63 /69 65
Fig. 3. 1H NMR spectra of the PHB recovered from the recombinant E. coli . The doublet, doublet of quadruplet and a multiplet at 1.3, 2.57 and 5.28
ppm are attributed to; the methyl group coupled to a proton, the methylene group adjacent to an asymmetric carbon atom bearing a single proton
and the methylene group, respectively. The signal at 7.25 ppm is characteristic of chloroform-d. Doublet of quadruplet of the methylene group is
indicative of the isotactic nature of PHB.
Table 1
PHB accumulation by the recombinant E. coli in basal medium
containing glucose (BMG), glycerol (BMGL), sucrose (BMS) or
sodium acetate (BMSA), and in LB medium
LB broth 1.090.5
BMG 4.091.2
BMGL 60.096.0
BMS 5.090.8
BMSA 4.091.1
a
Cell dry mass basis.
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