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Applied and Environmental Microbiology, January 2009, p. 29-38, Vol. 75, No. 1
0099-2240/09/$08.00+0     doi:10.1128/AEM.01344-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Biotechnological Process for Production of β-Dipeptides from Cyanophycin on a Technical Scale and Its Optimization {triangledown}

Ahmed Sallam, Alene Kast, Simon Przybilla, Tobias Meiswinkel, and Alexander Steinbüchel*

Institut für Molekulare Mikrobiologie und Biotechnologie, Westfälische Wilhelms-Universität Münster, D-48149 Münster, Germany

Received 16 June 2008/ Accepted 24 October 2008

A triphasic process was developed for the production of β dipeptides from cyanophycin (CGP) on a large scale. Phase I comprises an optimized acid extraction method for technical isolation of CGP from biomass. It yielded highly purified CGP consisting of aspartate, arginine, and a little lysine. Phase II comprises the fermentative production of an extracellular CGPase (CphEal) from Pseudomonas alcaligenes strain DIP1 on a 500-liter scale in mineral salts medium, with citrate as the sole carbon source and CGP as an inductor. During optimization, it was shown that 2 g liter–1 citrate, pH 6.5, and 37°C are ideal parameters for CphEal production. Maximum enzyme yields were obtained after induction in the presence of 50 mg liter–1 CGP or CGP dipeptides for 5 or 3 h, respectively. Aspartate at a concentration of 4 g liter–1 induced CphEal production with only about 30% efficiency in comparison to that with CGP. CphEal was purified utilizing its affinity for the substrate and its specific binding to CGP. CphEal turned out to be a serine protease with maximum activity at 50°C and at pH 7 to 8.5. Phase III comprises degradation of CGP to β-aspartate-arginine and β-aspartate-lysine dipeptides with a purity of over 99% (by thin-layer chromatography and high-performance liquid chromatography), employing a crude CphEal preparation. Optimum degradation parameters were 100 g liter–1 CGP, 10 g liter–1 crude CphEal powder, and 4 h of incubation at 50°C. The overall efficiency of phase III was 91%, while 78% (wt/wt) of the used CphEal powder with sustained activity toward CGP was recovered. The optimized process was performed with industrial materials and equipment and is applicable to any desired scale.

* Corresponding author. Mailing address: Institut für Molekulare Mikrobiologie und Biotechnologie, Westfälische Wilhelms-Universität Münster, D-48149 Münster, Germany. Phone: 49-251-8339821. Fax: 49-251-8338388. E-mail: steinbu{at}uni-muenster.de

{triangledown} Published ahead of print on 31 October 2008.

Applied and Environmental Microbiology, January 2009, p. 29-38, Vol. 75, No. 1
0099-2240/09/$08.00+0     doi:10.1128/AEM.01344-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Steinle, A., Bergander, K., Steinbuchel, A. (2009). Metabolic Engineering of Saccharomyces cerevisiae for Production of Novel Cyanophycins with an Extended Range of Constituent Amino Acids. Appl. Environ. Microbiol. 75: 3437-3446 [Abstract] [Full Text]  


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