Establishment of a simple and effective isolation method for cyanophycin from recombinant Saccharomyces cerevisiae

Abstract

An efficient, time-saving, and cost-effective method for isolation of the polyamide cyanophycin from recombinant Saccharomyces cerevisiae was established. Due to its simple procedure, this isolation method may be also applicable at industrial scale and also to other intracellular compounds in this yeast. Production of cyanophycin gained preferential interest in the past, as degradation products thereof are of pharmaceutical and technical interest. Recently, it was shown that Saccharomyces cerevisiae represents a putative candidate for cyanophycin synthesis at industrial scale. For identification of optimal isolation procedures, several parameters such as heat stress, freeze drying, and freeze/thaw cycles of transgenic yeast cells were compared for their effectiveness of cyanophycin isolation. Additionally, optimal resuspension solutions for the applied cells and minimal required materials or chemicals were determined to make the process most environmentally and economically friendly. Maximal cyanophycin granule polypeptide yields of 21% (w/w) were obtained after incubation of dry cells at 70 degrees C or 80 degrees C and precipitation of the polymer with two volumes of ethanol.

Increased Lysine Content Is the Main Characteristic of the Soluble Form of the Polyamide Cyanophycin Synthesized by Recombinant Escherichia coli

“…In addition, it was shown by SDS-PAGE that thermal treatment had no influence on the molecular mass of the isolated polymers. On the other hand, until now, no soluble CGP could be isolated from cells of recombinant E. coli by this method (19). An optimized and simplified protocol for the isolation of soluble CGP from E. coli based on the publications of Ziegler et al (7) and Steinle and Steinbüchel (19) is presented in this report.…”

“…Insoluble CGP from recombinant bacterial strains was always isolated from lyophilized cells to allow a conclusion about yield with regard to the CDM and to compare the productivities of different strains or different culture conditions. But the question of whether lyophilization of E. coli cells is necessary to improve the yield of soluble CGP in comparison to that of fresh cells, as it was demonstrated for yeasts (19), may be allowed. Furthermore, the omission of this step could reduce the period of downstream processing.…”

Stabilized and Immobilized Bacillus subtilis Arginase for the Biobased Production of Nitrogen‐Containing Chemicals

“…Heterologous expression in industrially relevant bacteria5–8 has opened up the possibility to produce CGP in considerable amounts. Even more interestingly, recent work on Saccharomyces cerevisiae 9,10 gives rise to possibilities for the simultaneous production of CGP and ethanol from amino acid‐rich waste streams from the agro‐industries 11–13…”

“…Since CGP is insoluble under physiological conditions, it can be easily isolated14–16 which, together with its defined composition, its integrated production and its high nitrogen content, make CGP an interesting feedstock for the production of nitrogen‐containing bulk chemicals 13…”

Material properties of the cyanobacterial reserve polymer multi-l-arginyl-poly-l-aspartate (cyanophycin)

“…Cyanophycin purification. Cyanophycin was purified using a procedure adapted from the literature [22,43]. Cell pellets were frozen at -80 °C, thawed, and resuspended in three volumes of 1.0 M hydrochloric acid for 4 hours at 30 °C with agitation.…”