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ÌâĿΪ¦Ã-Poly(glutamic acid)Formation by Bacillus licheniformishysiological and Biochemical Studies

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¦Ã-Poly(glutamic acid) formation by Bacillus licheniformis 9945a: physiological and biochemical studies
International Journal of Biological Macromolecules
Volume 16, Issue 5, 1994, Pages 265-275
doi:10.1016/0141-8130(94)90032-9
IDS Number: PU089
ISSN: 0141-8130
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abstract

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¦Ã-Poly(glutamic acid) formation by Bacillus licheniformis 9945a: physiological and biochemical studies

Gregory A. Birrer*, a, Anne-Marie Cromwicka and Richard A. Gross, a

aUniversity of Massachusetts Lowell, Department of Chemistry, One University Avenue, Lowell, MA 01854, USA

Received 8 March 1994;  revised 5 August 1994.  Available online 27 January 2003.

Abstract
Cryogenically frozen vegetative cells of Bacillus licheniformis 9945a derived from young mucoid colonies were used to inoculate ¦Ã-poly(glutamate) (¦Ã-PGA) production media containing l-glutamate, citrate and glycerol as carbon sources. A gel permeation chromatography (GPC) method was developed to determine ¦Ã-PGA volumetric yield and molecular weight directly using culture filtrates. For GPC volumetric yield measurements, a calibration curve was generated using purified ¦Ã-PGA to relate the ¦Ã-PGA GPC peak area and polymer weight. Purified ¦Ã-PGA was characterized by elemental analysis, 1H- and 13C-NMR spectroscopy. Cultures of B. licheniformis using all three carbon sources showed the following characteristics: cell growth mainly during the first 24 h; largest ¦Ã-PGA volumetric productivity (0.12 gl−1 h−1) between 48 and 96 h; 11 gl−1 ¦Ã-PGA volumetric yield by 96 h; reduction (utilization) of glycerol, glutamate and citrate during a 96 h cultivation time from 80 to 45 gl−1, 18 to 10 gl−1 and 12 to 1 gl−1, respectively; a decrease in pH from 7.4 to 5.5 by 42 h cultivation; acetic acid secretion into the medium at a maximum level of 4.5 gl−1 and detection of the metabolite 2,3-butanediol (as acetoin) as a fermentation by-product at 42 h and through a 96 h cultivation period. The presence of 2,3-butanediol indicated that the level of oxygen in the medium no longer supported a fully aerobic mode of metabolism. When the medium formulation was altered by removal of either citrate, l-glutamate or glycerol in shake flask experiments where pH was not controlled, 2.3, 9.0 and 4.0 gl−1, respectively, of ¦Ã-PGA were formed. Variation of the medium ionic strength by the addition of up to 4% (w/v) NaCl led to the formation of ¦Ã-PGA of relatively higher molecular weight but lower volumetric yield. Studies carried out on 5-day-old B. licheniformis cultures suggested that ¦Ã-PGA depolymerase is intracellularly located or cell-bound. Culture filtrates showed no significant ¦Ã-PGA depolymerase activity.

Keywords: Bacillus licheniformis 9945a; ¦Ã-poly(glutamate); gel permeation chromatography
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