Research Article: Production optimization of cyanophycinase ChpEal from Pseudomonas alcaligenes DIP1

Date Published: November 7, 2011

Publisher: Springer

Author(s): Ahmed Sallam, Dimitar Kalkandzhiev, Alexander Steinbüchel.

http://doi.org/10.1186/2191-0855-1-38

Abstract

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 serine-type 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.

Partial Text

Cyanophycin (cyanophycin granule polypeptide, CGP) is a naturally occurring poly(amino acid) that was first observed in cyanobacteria (Borzi 1887). It is accumulated in the early stationary growth phase (Mackerras et al. 1990; Liotenberg et al. 1996) and functions as storage compound for nitrogen, carbon, and energy (Elbahloul et al. 2005; Füser and Steinbüchel 2007). CGP consists of a poly(aspartic acid) backbone with arginine moieties linked to the β-carboxyl group of each aspartic acid by its α-amino group to form multi-L-arginyl-poly(L-aspartic acid) (Simon and Weathers 1976; Oppermann-Sanio and Steinbüchel 2003). CGP is accumulated intracellularly in the form of membraneless granules and is degraded by cells when growth is resumed (Allen and Weathers 1980).

Several extracellular cyanophycinases were identified in the last few years, and various applications have been proposed for the dipeptides produced by the action of these enzymes on CGP (Sallam and Steinbüchel 2010). A process for the production of dipeptides from CGP based on the cyanophycinase from P. alcaligenes strain DIP1 was described in previous studies (Sallam et al. 2009), but the identity of the cyanophycinase gene remained unknown. This study identified the cyanophycinase gene of this strain and optimized the production of the enzyme in recombinant E. coli strains.

The authors declare that they have no competing interests.

 

Source:

http://doi.org/10.1186/2191-0855-1-38

 

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