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Production optimization of cyanophycinase ChpEal from Pseudomonas alcaligenes DIP1

Pseudomonas alcaligenes DIP1 produces an extracellular cyanophycinase (CphEal). The corresponding gene (cphEal) was identified from subclones of a genomic DNA gene library by heterologously expressing the functionally active enzyme in Escherichia coli. The nucleotide sequence of the gene (1260 base pairs) was determined indicating a theoretical mass of 43.6 kDa (mature CphEal) plus a leader peptide of 2,6 kDa which corresponds well to the apparent molecular mass of 45 kDa as revealed by SDS-PAGE. The enzyme exhibited a high sequence identity of 91% with the extracellular cyanophycinase from P. anguilliseptica strain BI and carried an N-terminal Sec secretion signal peptide. Analysis of the amino acid sequence of cphE revealed a putative catalytic triad consisting of the serine motif GXSXG plus a histidine and a glutamate residue, suggesting a catalytic mechanism similar to serinetype proteases. The cyanophycinase (CphEal) was heterologously produced in two different E. coli strains (Top10 and BL21(DE3)) from two plasmid vectors (pBBR1MCS-4 and pET-23a(+)). The signal peptide of CphEal was cleaved in E. coli, suggesting active export of the protein at least to the periplasm. Substantial enzyme activity was also present in the culture supernatants. The extracellular cyanophycinase activities in E. coli were higher than activities in the wild type P. alcaligenes DIP1 in complex LB medium. Highest extracellular enzyme production was achieved with E. coli BL21(DE3) expressing CphEal from pBBR1MCS-4. Using M9 minimal medium was less effective, but the relatively low cost of mineral salt media makes these results important for the industrial-scale production of dipeptides from cyanophycin.

Titel: Production optimization of cyanophycinase ChpEal from Pseudomonas alcaligenes DIP1
Verfasser: Sallam, Ahmed GND
Kalkandzhiev, Dimitar
Steinbüchel, Alexander GND
Organisation: FB 13: Biologie
Dokumenttyp: Artikel
Medientyp: Text
Erscheinungsdatum: 07.11.2011
Publikation in MIAMI: 14.02.2013
Datum der letzten Änderung: 25.08.2014
Quelle: AMB Express 1 (2011) 38
Schlagworte: Cyanophycinase; Cyanophycin degradation; Pseudomonas alcaligenes
Fachgebiete: Biowissenschaften; Biologie
Lizenz: CC BY 2.0
Sprache: Englisch
Anmerkungen: Finanziert durch den Open-Access-Publikationsfonds 2011/2012 der Deutschen Forschungsgemeinschaft (DFG) und der Westfälischen Wilhelms-Universität Münster (WWU Münster).
Format: PDF-Dokument
URN: urn:nbn:de:hbz:6-47389392775
DOI: 10.1186/2191-0855-1-38